Research in the Guikema laboratory is funded by the Innovational Research Incentives Scheme from The Netherlands Organization for Scientific Research (VIDI grant 016126355) and the 'Stichting Kinderen Kankervrij' KiKA (project 252).

1. Treatment of Cells and DNA Damage Induction
<ol>
	<li>Culture the human BCR-ABL+ B-cell acute lymphoblastic cell lines BV173 or SUP-B15 in IMDM supplemented with 20% FCS, 50 &#956;M &#946;-mercaptoethanol, 2 mM L-glutamine, 100 U/mL penicillin and 100 &#956;g/mL streptomycin at 37 &#176;C in an atmosphere of 5% CO2. Count cells and plate at 2 x 106 cell/mL in 6-wells plates, at 5 mL/well. 
	NOTE: Optionally, suspension cell lines of various origin can used.</li>
	<li>To arrest the BCR-ABL+ B-ALL cells in the G1-phase of the cell-cycle, add 5 &#956;M of imatinib methanesulfonate (STI571), and incubate overnight.
	<ol>
		<li>Optionally, omit this step when studying protein-protein interactions throughout the cell-cycle or in BCR-ABL-negative cell types.</li>
	</ol>
	</li>
	<li>Induce DNA damage by adding 50 ng/mL neocarzinostatin (NCS) to the culture, and incubate for 2 h.
	<ol>
		<li>Alternatively, induce DNA damage by irradiating the cells using a radioactive source [137Cs] at 0.5 Gy/min, at a dose of 5 Gy. After irradiation, let the cell recover for 2 h at 37 &#176;C in an atmosphere of 5% CO2.</li>
	</ol>
	</li>
	<li>Optionally, to assess the involvement of ATM kinase activity in response to DNA damage, add 5 &#956;M of the ATM kinase inhibitor KU55933 prior to addition of NCS or the irradiation treatment.</li>
	<li>Prepare 1x PBS + 10% BSA by layering 5 g of fraction-V BSA on top of 50 mL of 1x PBS in a beaker or Erlenmeyer flask, and let sit at RT until BSA is dissolved. Do not shake or stir as this will cause extensive foaming. Slowly filter through a 0.22 &#956;m filter and keep on ice.</li>
	<li>Harvest cells and wash twice with ice-cold 1x PBS. Count the cells and resuspend at 2 x 106/mL in 1x PBS + 10% BSA. Keep cells on ice.</li>
</ol>
2. Cytocentrifugation, Fixation and Permeabilization
<ol>
	<li>Prepare the 4% paraformaldehyde solution in 1x PBS freshly. Add 2 g of PFA to 50 mL of 1x PBS and incubate in a 55 &#176;C shaking water bath until the solution is clear. Filter the solution through a 0.22 &#956;m filter and store at RT until use. 
	NOTE: Optionally, 4% PFA fixative can be stored frozen at -20 &#176;C. After freezing, thaw only once for use. Otherwise, the 4% PFA solution in PBS can be purchased directly.</li>
	<li>Carefully polish 76 mm x 26 mm microscopy slides with lint-free tissue and degrease them by placing in 96% ethanol in a Coplin jar for 5 min. Let the microscopy slides air-dry for 10 min.</li>
	<li>Assemble microscopy slides into the cytospin/cytology funnels with an area of 6 mm. Pre-coat slides by pipetting 50 &#956;L of 1x PBS + 10% BSA into each funnel and centrifuge for 1 min at 500 rpm.</li>
	<li>Add 100 &#956;L of the cell-suspension (equaling 0.2 x 106 cells) to each funnel and centrifuge for 5 min at 500 rpm. For each antibody combination, at least 4 preparations are required (dual-antibody experiment; two single-antibody controls; no antibodies control).</li>
	<li>Carefully disassemble the funnels, and make sure not to touch the spots. Proceed to sample fixation immediately.
	<ol>
		<li>Optionally, let slides air-dry for at least 30 min. After air-drying, wrap slides individually in aluminum foil and store at -20 &#176;C until use. When using frozen preparations, make sure to thaw the slides wrapped in a paper towel to prevent overt condensation onto the slides.</li>
	</ol>
	</li>
	<li>Draw a circle (approximate diameter 15-20 mm) around the spot using a PAP pen liquid blocker (5 mm tip).</li>
	<li>Add 50 &#956;L of 4% PFA fixation solution to each spot and incubate for 30 min at RT.</li>
	<li>Wash the cells 3 times for 5 min with 1x PBS in a Coplin jar at RT with gentle agitation.</li>
	<li>Dry the slides around the spots with a lint-free tissue and remove as much liquid from the spot as possible using a pipet without touching the spot, or by tilting the slide. Add 50 &#956;L of 0.25% Triton-X in 1x PBS to each spot and incubate for 10 min at RT without agitation.</li>
	<li>Wash the slides 3 times for 5 min in ~50-60 mL of 0.05% Tween-20 in TBS (TBS-T) in a Coplin jar at RT with gentle agitation. 10 slides can be processed simultaneously in this fashion.</li>
</ol>
3. Blocking and Primary Antibody Incubation
<ol>
	<li>Tap off TBS-T and dry the slides around the spots with a lint-free tissue. Remove as much liquid from the spot as possible using a pipet without touching the spot, or by tilting the slide. Shortly vortex the blocking solution and add one drop (~ 40 &#956;L) to each spot. Incubate for 1 h at 37 &#176;C in a pre-warmed humidified chamber.</li>
	<li>Prepare the antibody cocktail by mixing the goat-anti-ATM at 1:1,000 and the rabbit-anti-phospho-Ser15-p53 at 1:100 in commercial antibody diluent, vortex antibody diluent before use. For control experiments, prepare antibody dilutions containing only the goat-anti-ATM and only the rabbit-anti-phospho-Ser15-p53 antibody (single-antibody controls).</li>
	<li>Tap off the blocking solution but take care not to let the cells dry prior to adding the antibody dilutions. Add 30 &#956;L of each antibody cocktail dilution and incubate in a humidified chamber overnight at 4 &#176;C.</li>
	<li>Prepare in situ Wash Buffer A and Wash Buffer B by dissolving the content of one pouch of Buffer A and Buffer B mix in a final volume of 1,000 mL of water each.
	<ol>
		<li>Alternatively, prepare wash buffer A by dissolving 8.8 g of NaCl, 1.2 g of Tris-base and 0.5 mL of Tween-20 in 800 mL water. Adjust pH to 7.4 using HCl and add to a final volume of 1,000 mL.</li>
		<li>Alternatively, prepare Wash Buffer B by dissolving 5.84 g of NaCl, 4.24 of Tris-base and 26.0 g of Tris-HCl in 500 mL of water. Adjust pH to 7.5 using HCl. Add water to a final volume of 1,000 mL.</li>
		<li>Filter both solutions through a 0.22 &#956;m filter.</li>
	</ol>
	</li>
	<li>Wash the slides 2 times for 5 min with 1x in situ wash buffer A in a Coplin jar at RT with gentle agitation.</li>
</ol>
4. PLA Probes, Ligation and Amplification
<ol>
	<li>Prepare PLA probes by diluting the anti-goat PLUS and anti-rabbit MINUS both 1:5 in the commercial antibody diluent. Tap off 1x in situ wash buffer A from the slides and add 30 &#956;L of the PLA probe-mixture to each spot. Incubate 1 h at 37 &#176;C in a pre-warmed humidified chamber. 
	NOTE: Any combination of secondary antibodies PLUS and MINUS PLA probes can be used (anti-mouse, anti-rabbit, anti-goat), as long as the applied primary antibodies are raised in different species, and the combination of PLA probes should always contain a PLUS probe and a MINUS probe.</li>
	<li>Wash slides 2 times for 5 min with ~50-60 mL 1x in situ wash buffer A in a Coplin jar at RT with gentle agitation.</li>
	<li>Prepare the Ligation-Ligase solution on ice by adding 1 &#956;L of Ligase to 39 &#956;L of Ligation solution and add 40 &#956;L of Ligation-Ligase solution to each spot. Incubate 30 min at 37 &#176;C in a pre-warmed humidified chamber.</li>
	<li>Wash the slides 2 times for 5 min with ~50-60 mL 1x in situ wash buffer A in a Coplin jar at RT with gentle agitation.</li>
	<li>Prepare the Amplification-Polymerase mixture on ice by diluting the Amplification stock solution 1:5 in water. Then add 0.5 &#956;L of polymerase to 39.5 &#956;L of 1x Amplification solution for each spot. 
	NOTE: The amplification reagents are light sensitive and should be protected against direct exposure to light. The amplification reagent comes in four different colors (RED: excitation 594 nm, emission 624 nm; FarRED: excitation 644 nm, emission 669 nm; ORANGE: excitation 554 nm, emission 576 nm; GREEN: excitation 501 nm, emission 523 nm), make sure that the microscopy setup available has suitable excitation properties and filters to image these colors.</li>
	<li>Tap off 1x in situ wash buffer A and add 40 &#956;L of Amplification-Polymerase mixture per spot. Incubate for 100 min at 37 &#176;C in a pre-warmed humidified chamber.</li>
</ol>
5. Mounting and Imaging
<ol>
	<li>Prepare 0.01x in situ wash buffer B by adding 1 mL of 1x Wash Buffer B to 99 mL of water.</li>
	<li>Tap off the Amplification-Polymerase mixture and wash slides 2 times for 10 min with 1x in situ wash buffer B in a Coplin jar at RT with gentle agitation.</li>
	<li>Wash the slides 1 min in 0.01x wash buffer B.</li>
	<li>Tap off excess 0.01x wash buffer B and dry the slides around the spots with a lint-free tissue. Remove as much liquid from the spot as possible using a pipet without touching the spot, or by tilting the slide.</li>
	<li>Dilute DAPI-containing mounting medium 1:5 in mounting medium without DAPI to avoid overstaining of the nuclei.</li>
	<li>Add 25-30 &#956;L of 1:5 DAPI-containing mounting medium to a 24 mm x 24 mm coverslip and pick up the cover slip with the slide, apply slight pressure to ensure that there are no trapped air bubbles. Seal the coverslip with nail polish and wait at least 15 min before imaging.</li>
	<li>Analyze the slides using a (confocal) fluorescence microscope.
	<ol>
		<li>Count the number of punctate PLA signals per cell in at least 4 acquired image fields (20x objective, counting at least 20 cells per condition or antibody combination, based on a power calculation using an anticipated mean of 7 &#177; 5 signals in the treated cells, and 2 &#177; 2 signals in the untreated cells, with a probability of a type-I error of 0.05, and a power of 80%, as reported previously15). 
		NOTE: The best resolution is achieved by acquisition of a Z-stack and deconvolution using software packages (e.g., ImageJ, the Leica Acquisition Suite (LAS)). The signals should be readily discernible within the nuclear perimeter, as defined by the DAPI staining.</li>
		<li>Do not count signals that clearly appear outside the nuclei. Typically, NCS treatment or &#947;-irradiation results in approximately 5-10 phosho-p53/ATM PLA signals per cell. The &#39;single-antibody&#39; controls may give some background signal, but this should not exceed 1-2 PLA signals per 1 out of every 20 cells.</li>
		<li>For publication and presentation purposes, capture images using a 40X or 63X immersion oil objective. Slides can be stored at 4 &#176;C and should be protected from exposure to light.</li>
	</ol>
	</li>
</ol>

<ol>
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D., Deng, C. X., Tagle, D. A., Wynshaw-Boris, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atm+selectively+regulates+distinct+p53-dependent+cell-cycle+checkpoint+and+apoptotic+pathways.">Atm selectively regulates distinct p53-dependent cell-cycle checkpoint and apoptotic pathways.</a> Nature Genet. 17 (4), 453-456 (1997).</li><li>Tibbetts, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+ATR+in+the+DNA+damage-induced+phosphorylation+of+p53.">A role for ATR in the DNA damage-induced phosphorylation of p53.</a> Genes Dev. 13 (2), 152-157 (1999).</li><li>Schermelleh, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subdiffraction+Multicolor+Imaging+of+the+Nuclear+Periphery+with+3D+Structured+Illumination+Microscopy.">Subdiffraction Multicolor Imaging of the Nuclear Periphery with 3D Structured Illumination Microscopy.</a> Science. 320 (5881), 1332-1336 (2008).</li><li>Chapman, J. R., Sossick, A. J., Boulton, S. J., Jackson, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BRCA1-associated+exclusion+of+53BP1+from+DNA+damage+sites+underlies+temporal+control+of+DNA+repair.">BRCA1-associated exclusion of 53BP1 from DNA damage sites underlies temporal control of DNA repair.</a> J Cell Sci. 125 (Pt 15), 3529-3534 (2012).</li><li>Gullberg, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sense+of+closeness:+protein+detection+by+proximity+ligation.">A sense of closeness: protein detection by proximity ligation.</a> Curr Opin Biotechnol. 14 (1), 82-86 (2003).</li><li>S&#246;derberg, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+proteins+and+their+interactions+in+cells+and+tissues+using+the+in+situ+proximity+ligation+assay.">Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay.</a> Methods. 45 (3), 227-232 (2008).</li><li>Dong, Y., Shannon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneous+immunosensing+using+antigen+and+antibody+monolayers+on+gold+surfaces+with+electrochemical+and+scanning+probe+detection.">Heterogeneous immunosensing using antigen and antibody monolayers on gold surfaces with electrochemical and scanning probe detection.</a> Anal Chem. 72 (11), 2371-2376 (2000).</li><li>Jackson, S. P., Bartek, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DNA-damage+response+in+human+biology+and+disease.">The DNA-damage response in human biology and disease.</a> Nature. 461 (7267), 1071-1078 (2009).</li><li>Economopoulou, P., Pappa, V., Papageorgiou, S., Dervenoulas, J., Economopoulos, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormalities+of+DNA+repair+mechanisms+in+common+hematological+malignancies.">Abnormalities of DNA repair mechanisms in common hematological malignancies.</a> Leuk Lymphoma. 52 (4), 567-582 (2011).</li><li>Rendleman, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+variation+in+DNA+repair+pathways+and+risk+of+non-Hodgkin's+lymphoma.">Genetic variation in DNA repair pathways and risk of non-Hodgkin's lymphoma.</a> PloS One. 9 (7), e101685 (2014).</li><li>Ochodnicka-Mackovicova, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DNA+Damage+Response+Regulates+RAG1/2+Expression+in+Pre-B+Cells+through+ATM-FOXO1+Signaling.">The DNA Damage Response Regulates RAG1/2 Expression in Pre-B Cells through ATM-FOXO1 Signaling.</a> J Immunol. , (2016).</li><li>Banin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+phosphorylation+of+p53+by+ATM+in+response+to+DNA+damage.">Enhanced phosphorylation of p53 by ATM in response to DNA damage.</a> Science. 281 (5383), 1674-1677 (1998).</li></ol>

The authors have nothing to disclose.

In this report, it is demonstrated that PLA can be used to determine and visualize the specific interaction between proteins in suspension cell cultures. Of note, the protocol described here is not restricted to the study of DNA repair complexes but also applies to visualize and quantify other protein-protein interactions in suspension cell cultures. It is shown that the ATM kinase interacts with phosphorylated p53 in G1-arrested BCR-ABL+ B-ALL cells when exposed to a DNA damage-inducing agent. Previously, the PLA techni...

DNA damage triggers a highly regulated series of events involving protein-protein interactions and post-translational modifications that ensures the efficient and rapid repair of DNA, thereby safeguarding genomic integrity1. Typically, DNA repair is studied in cells exposed to ionizing radiation by monitoring the formation of so-called ionizing radiation-induced foci (IRIF) by (confocal) fluorescence microscopy. Many DNA repair and DNA damage-sensing proteins form IRIFs, which represent protein complexes that nucleate at chromatin sites sustaining DNA damage2,3. The location and resolution of IRIFs over time gives important insight into spatiotemporal organization of DNA repair, and may indicate the involvement of different DNA repair pathways. The nature of the DNA damage and the cell-cycle stage in which the damage is attained determines which DNA repair pathway is activated. For instance, in cells actively engaged in DNA replication (S-phase), homologous recombination (HR) is the dominant DNA repair pathway, whereas in cells in the G1- or G2/M-phase of the cell-cycle, the non-homologous end-joining (NHEJ) repair pathway predominates. One of the earliest events following DNA damage is the activation of the DNA damage-sensing kinases Ataxia Telangiectasia-Mutated protein (ATM), which is mostly active in the G1- and G2/M-phases of the cell-cycle and regulates NHEJ, and Ataxia Telangiectasia and Rad3-related protein (ATR), which acts in S-phase by activating HR. Both ATM and ATR are very pleiotropic kinases that phosphorylate many proteins that are involved in DNA repair, cell death and survival4. Both kinases have been shown to phosphorylate and activate the tumor-suppressor protein p53 following the exposure to genotoxic stress, indicating that these kinases are upstream mediators of a pivotal tumor suppressive signaling axis5,6.
The formation and composition of IRIFs is typically assessed by determining co-localization of different proteins using dual-color immunofluorescence staining and microscopy, however, not all proteins that are part of repair protein complexes form IRIFs, which limits the applicability of this approach. Moreover, (confocal) immunofluorescence microscopy is limited by the diffraction properties of light, resulting in a rather poor spatial resolution of about 200-300 nm, exceeding the size of most subcellular structures, which essentially prohibits the direct interrogation of protein-protein interactions at the molecular level. As such, the co-localization of immunofluorescence staining patterns as detected by (confocal) fluorescence microscopy is not necessarily indicative for direct protein-protein interactions. Recently, new super-resolution technologies have been developed, such as three-dimensional structured illumination microscopy (3D-SIM)7, which was successfully used to study 53BP1 and BRCA1 IRIF formation at nano-scale detail, revealing the spatial distribution characteristics of these proteins that could not detected by confocal laser scanning microscopy8.
Several other methods can be used to detect protein-protein interactions in vivo, such as co-immunoprecipitation, pull-down methods and yeast two-hybrid screening approaches. However, these techniques are rather cumbersome, require large amounts of cells or proteins or involve overexpression of proteins, which introduces experimental artifacts. More recently, a novel technique has been developed that allows the visualization and quantification of protein-protein interactions in situ (i.e. in cells and in tissues), which is termed Proximity Ligation Assay (PLA)9,10. Primary antibodies that recognize two proteins of interest are detected by secondary antibodies that are conjugated to oligonucleotides (so-called PLA probes). If the two different secondary antibodies are sufficiently close due to interactions between the proteins recognized by the primary antibodies, the conjugated oligonucleotides hybridize and can be ligated to form a closed circular DNA substrate. This circular substrate is subsequently amplified by rolling circle amplification, and visualized with fluorochrome-conjugated complementary oligonucleotides. Using PLA, the subcellular localization of the protein-protein interaction is preserved as the fluorescently labeled rolling circle amplification-product remains attached to the PLA probes. The resolution of this assay is &#60;50 nm, based on the finding that the diameter of an antibody is approximately 7-10 nm11. Rolling circle amplification can only take place in case two pairs of antibodies (primary + secondary) physically interact within the perimeter that is defined by their size (10 + 10 + 10 + 10 = 40 nm). The signal amplification step increases the sensitivity of the PLA assay and enables the detection of interactions of scarcely expressed proteins. PLA generates punctate, foci-like signals patterns that can be quantified on a per cell basis, by which the intra- and inter-cellular variation in protein-protein interactions can be assessed.
The formation and composition of DNA repair complexes and IRIFs is mostly studied in adherent cell lines such as the human bone osteosarcoma epithelial cell line U2OS, the human embryonic kidney cell line HEK293 and the retinal pigment epithelial cell line RPE-1, which are fast-growing and easy to transfect. Suspension cell cultures such a lymphoid and myeloid cell lines are used less frequently, as these are less amenable to transfection and generally do not adhere to coverslips, thus requiring additional/alternative steps for imaging. The resolution of DNA damage is however very relevant in the context of lymphoid and myeloid malignancies, as the DNA damage response is frequently affected by genomic (driver) aberrations in these tumors, playing a pivotal role in the malignant transformation of normal lymphoid and myeloid (progenitor) cells12,13,14.
This protocol describes how PLA can be used to assess and quantify protein-protein interactions following the induction of DNA damage in suspension cell cultures. Here, PLA is performed to determine and visualize the interactions between ATM and p53 upon DNA damage in human B-cell leukemia cells that are induced to undergo a G1-phase cell-cycle arrest. Of note, the protocol presented here is not restricted to studying ATM and p53 interactions in G1-arrested leukemia cells, but can also be used to visualize other protein-protein interactions in various cell types and suspension cell cultures.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BV173 cell line</td>
			<td>DSMZ</td>
			<td>AC-20</td>
			<td>BCR-ABL+ B-ALL cell line</td>
		</tr>
		<tr>
			<td>SUP-B15 cell line</td>
			<td>DSMZ</td>
			<td>ACC-389</td>
			<td>BCR-ABL+ B-ALL cell line</td>
		</tr>
		<tr>
			<td>Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM)</td>
			<td>Gibco (Life Technologies)</td>
			<td>21980-032</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Calf Serum</td>
			<td>Sigma Aldrich</td>
			<td>F7524</td>
			<td>lot #: 064M3396</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco (Life Technologies)</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin/streptomycin</td>
			<td>Gibco (Life Technologies)</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>imatinib methanesulfonate</td>
			<td>LC Laboratories</td>
			<td>I-5508</td>
			<td>Dissolve in DMSO, prepare 10 mM stock solution</td>
		</tr>
		<tr>
			<td>neocarzinostatin</td>
			<td>Sigma Aldrich</td>
			<td>N9162</td>
			<td>Mutagenic/teratogenic, handle with care</td>
		</tr>
		<tr>
			<td>KU55933</td>
			<td>Selleckchem</td>
			<td>S1092</td>
			<td>Dissolve in DMSO, prepare 5 mM stock solution</td>
		</tr>
		<tr>
			<td>Starfrost Microscopy Slides</td>
			<td>Waldemar Knittel</td>
			<td>VA11200 003FKB</td>
			<td></td>
		</tr>
		<tr>
			<td>PAP pen liquid blocker</td>
			<td>Sigma Aldrich</td>
			<td>Z377821-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytospin funnel</td>
			<td>Q Path Labonord SAS</td>
			<td>003411324</td>
			<td></td>
		</tr>
		<tr>
			<td>Duolink In Situ Red Starter Kit Goat/Rabbit</td>
			<td>Sigma Aldrich</td>
			<td>DUO92105</td>
			<td>Available for different species/combinations, also available in FarRED, Orange and Green</td>
		</tr>
		<tr>
			<td>goat-anti-ATM</td>
			<td>Bethyl Laboratories</td>
			<td>A300-136A</td>
			<td>PLA-grade; we succesfully used lot#A300-136A-1 in our studies</td>
		</tr>
		<tr>
			<td>rabbit-anti-phospho-Ser15-p53</td>
			<td>Cell Signaling Technology</td>
			<td>9284</td>
			<td>We succesfully used lot #9284-4 in our studies</td>
		</tr>
		<tr>
			<td>Vectashield antifading mounting medium with DAPI</td>
			<td>Vector Labs</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield antifading mounting medium</td>
			<td>Vector Labs</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>4% paraformaldehyde in PBS</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-281692</td>
			<td>Also available from various other vendors</td>
		</tr>
	</tbody>
</table>

detection and visualization of dna damage-induced protein complexes in suspension cell cultures using the proximity ligation assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Phosphorylation of p53 at residue Ser15 was shown to be dependent on ATM kinase activity16. To demonstrate and confirm the specificity of the PLA technique on cytospin preparations of suspension cell cultures, it is shown that induction of DNA damage by 2 h NCS treatment of BCR-ABL+ B-ALL cells arrested in the G1-phase of the cell-cycle resulted in the specific interaction between ATM and phospho-Ser15-p53, as expected. Punctate PLA signals were observed in the nuc...

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Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

detection-visualization-dna-damage-induced-protein-complexes

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

9.9K Views.  Academic Medical Center, University of Amsterdam, Lymphoma and Myeloma Center Amsterdam (LYMMCARE). Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress. 

Video: Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation is a popular technique to detect protein-protein interactions. Subsequent Western blot analysis is the most common method to visualize co-immunoprecipitated proteins. Recently, the proximity ligation assay has become a powerful tool to visualize protein-protein interactions in situ and provides the possibility to quantify protein-protein interactions by this method. Similar to conventional immunocytochemistry, the proximity ligation assay technique is also based on the accessibility of primary antibodies to the antigens, but in contrast, proximity ligation assay detects protein-protein interactions with a unique technique involving rolling-circle PCR, while conventional immunocytochemistry only shows co-localization of proteins.
Nuclear factor 90 (NF90) and RNA-binding motif protein 3 (RBM3) have been previously demonstrated as interacting partners. They are predominantly localized in the nucleus, but also migrate into the cytoplasm and regulate signaling pathways in the cytoplasmic compartment. Here, we compared NF90-RBM3 interaction in both the nucleus and the cytoplasm by co-immunoprecipitation and proximity ligation assay. In addition, we discussed the advantages and limitations of these two techniques in visualizing protein-protein interactions in respect to spatial distribution and the properties of protein-protein interactions.

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions can occur in both the nucleus and the cytoplasm of a cell. To investigate these interactions, traditional co-immunoprecipitation and modern proximity ligation assay are applied. In this study, we compare these two methods to visualize the distribution of NF90-RBM3 interactions in the nucleus and the cytoplasm.

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation ...

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of RNA-protein interactions. These interactions have been traditionally analyzed with polyacrylamide gels generated between two glass plates and samples electrophoresed vertically. However, polyacrylamide gels cast in trays and electrophoresed horizontally offers several advantages. For example, horizontal gels used to analyze complexes between fluorescent RNA substrates and specific proteins can be imaged multiple times as electrophoresis progresses. This provides the unique opportunity to monitor RNA-protein complexes at several points during the experiment. In addition, horizontal gel electrophoresis makes it possible to analyze many samples in parallel. This can greatly facilitate time course experiments as well as analyzing multiple reactions simultaneously to compare different components and conditions. Here we provide a detailed protocol for generating and using horizontal native gel electrophoresis for analyzing RNA-Protein interactions.

Native polyacrylamide gel electrophoresis is a fundamental tool for analyzing RNA-protein interactions. Traditionally most experiments have used vertical gels. However, horizontal gels provide several advantages, such as the opportunity to monitor complexes during electrophoresis. We provide a detailed protocol for generating and using horizontal native gel electrophoresis.

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of ...

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of diverse cellular functions, including cytoskeletal dynamics, cell motility, cell polarity, axonal guidance, vesicular trafficking, and cell cycle control. Changes in Rho GTPase signaling play an essential regulatory role in many pathological conditions, such as&#160;cancer, central nervous system diseases, and immune system-dependent diseases. The posttranslational modification of Rho GTPases (i.e., prenylation by mevalonate pathway intermediates) and GTP binding are key factors which affect the activation of this protein. In this paper, two essential and simple methods are provided to detect a broad range of Rho GTPase prenylation and GTP binding activities. Details of the technical procedures that have been used are explained step by step in this manuscript.

Here we describe a protocol to investigate the prenylation and guanosine-5'-triphosphate (GTP)-loading of Rho GTPase. This protocol consists of two detailed methods, namely membrane fractionation and a GTPase-linked immunosorbent assay. The protocol can be used for measuring the prenylation and GTP loading of different other small GTPases.

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in understanding how such pathways are organized and how they function. There are many methods to detect protein-protein interactions in cells, but relatively few can be used to detect interactions between endogenous proteins. One such method, the proximity ligation assay (PLA), has several advantages to recommend its use. Compared to other common methods of protein-protein interaction analysis, PLA has relatively high sensitivity and specificity, can be performed with minimal cell manipulation, and, in the protocol described herein, requires only two target-specific antibodies derived from different species (e.g., from mouse and rabbit) and one specialized reagent: a set of secondary antibodies that are covalently linked to specific oligonucleotides that, when brought in close proximity of one another, create an amplifiable platform for in situ PCR or rolling circle amplification. In this presentation, we show how to apply the PLA technique to visualize changes in MST1 and MST2 proximity in fixed cells. The technique described in this manuscript is particularly applicable for the analysis of cell signaling studies.

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Here, we show how to use a Proximity Ligation Assay (PLA) to visualize MST1/MST2 heterodimerization in fixed cells with high sensitivity.

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in ...

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have developed a simple non-labeled and non-radio-isotopic method using a matrix-assisted laser desorption ionization with time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis for a proofreading study. Here, a DNA polymerase [e.g., the Klenow fragment (KF) of Escherichia coli DNA polymerase I (pol I) in this study] in the presence of all four dideoxyribonucleotide triphosphates is used to process a mismatched primer-template duplex. The mismatched primer is then proofread/extended and subjected to MALDI-TOF MS. The products are distinguished by the mass change of the primer down to single nucleotide variations. Importantly, a proofreading can also be determined for internal single mismatches, albeit at different efficiencies. Mismatches located at 2-4-nucleotides (nt) from the 3' end were efficiently proofread by pol I, and a mismatch at 5 nt from the primer terminus showed only a partial correction. No proofreading occurred for internal mismatches located at 6 - 9 nt from the primer 3' end. This method can also be applied to DNA repair assays (e.g., assessing a base-lesion repair of substrates for the endo V repair pathway). Primers containing 3' penultimate deoxyinosine (dI) lesions could be corrected by pol I. Indeed, penultimate T-I, G-I, and A-I substrates had their last 2 dI-containing nucleotides excised by pol I before adding a correct ddN 5'-monophosphate (ddNMP) while penultimate C-I mismatches were tolerated by pol I, allowing the primer to be extended without repair, demonstrating the sensitivity and resolution of the MS assay to measure DNA repair.

A non-labeled, non-radio-isotopic method for DNA polymerase proofreading and a DNA repair assay was developed by using high-resolution MALDI-TOF mass spectrometry and a single nucleotide extension strategy. The assay proved to be very specific, simple, rapid, and easy to perform for proofreading and repair patches shorter than 9-nucleotides.

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have ...

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the translocon at the outer (TOC) and inner (TIC) chloroplast membranes. The TOC and TIC complexes are multimeric and probably contain yet unknown components. One of the main goals in the field is to establish the complete inventory of TOC and TIC components. For the isolation of TOC-TIC complexes and the identification of new components, the preprotein receptor TOC159 has been modified N-terminally by the addition of the tandem affinity purification (TAP) tag resulting in TAP-TOC159. The TAP-tag is designed for two sequential affinity purification steps (hence "tandem affinity"). The TAP-tag used in these studies consists of a N-terminal IgG-binding domain derived from Staphylococcus aureus Protein A (ProtA) followed by a calmodulin-binding peptide (CBP). Between these two affinity tags, a tobacco etch virus (TEV) protease cleavage site has been included. Therefore, TEV protease can be used for gentle elution of TOC159-containing complexes after binding to IgG beads. In the protocol presented here, the second Calmodulin-affinity purification step was omitted. The purification protocol starts with the preparation and solubilization of total cellular membranes. After the detergent-treatment, the solubilized membrane proteins are incubated with IgG beads for the immunoisolation of TAP-TOC159-containing complexes. Upon binding and extensive washing, TAP-TOC159 containing complexes are cleaved and released from the IgG beads using the TEV protease whereby the S. aureus IgG-binding domain is removed. Western blotting of the isolated TOC159-containing complexes can be used to confirm the presence of known or suspected TOC and TIC proteins. More importantly, the TOC159-containing complexes have been used successfully to identify new components of the TOC and TIC complexes by mass spectrometry. The protocol that we present potentially allows the efficient isolation of any membrane-bound protein complex to be used for the identification of yet unknown components by mass spectrometry.

We here present a proven and tested protocol for the purification of chloroplast protein import complexes (TOC-TIC complex) using the TAP-tag. The one-step affinity-isolation protocol can potentially be applied to any protein and be used to identify new interaction partners by mass spectrometry.

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

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Chapters in this video

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and GTPase-linked Immunosorbent Assay

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves