Rabbit anti xrcc5

To detect phosphorylated interacting proteins, we performed immunoprecipitation experiments using mouse anti-phospho (p)-serine (Sigma) or mouse anti-phospho (p)-threonine antibodies (Santa Cruz), as previously described (Lee et al., 2015a (link)). Briefly, transfected cells were suspended in 180 μl lysis buffer and 20 μl 10% SDS (final concentration of SDS, 1%). The samples were boiled for 10 min to denature proteins. Then, 1.8 ml of lysis buffer containing protease and phosphatase inhibitors was added to the samples (final concentration of SDS, 0.1%). The lysates were immunoprecipitated overnight 4°C with 2 μl of anti-p-serine or anti-p-threonine antibody, and 30 μl of protein G agarose beads were added and incubated for an additional 2 h. The beads were washed three times with lysis buffer, and the precipitated proteins were eluted in Laemmli 2× sample buffer (Sigma) at 100°C for 5 min. Phosphorylated interacting candidates were detected by immunoblotting using rabbit cyclin B1 (Cell signaling) and rabbit anti-XRCC5 (Santa Cruz) antibodies.

Co-immunoprecipitation samples were subjected to SDS-PAGE on acrylamide gel. For Commassie blue staining, the acrylamide gel was stained with Brilliant Blue R250 Protein solution (Elpis, Korea) for 1 h with gentle agitation and then washed three times with destaining solution containing 20% methanol and 10% acetic acid for 1 h. For Western blotting, SDS-PAGE gel was transferred onto PVDF membranes (Millipore). After blocking with 5% skimmed milk in TBS-T buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween20) for 1 h, membranes were incubated with the primary antibodies at 4°C with shaking overnight. After incubated with the secondary antibody for 2 h, immunocomplexes were detected by use of the femto, Pico or ECL reagent (Thermo). Antibodies for Western blotting are from the companies as follows: mouse/rabbit anti-flag antibody (Sigma), mouse anti-VRK1 (Santa Cruz), rabbit anti-VRK3 (Sigma), mouse ant-ALY antibody (Santa Cruz), rabbit anti-hsp70 (Origene), rabbit anti-XRCC5 (Santa Cruz), rabbit anti-B23 antibody (Santa Cruz), rabbit anti-C23 antibody (Santa Cruz), rabbit anti-PARP1 antibody (Cell signaling), rabbit anti-cyclin B1 (Santa Cruz), rabbit anti-p53 (Cell signaling) and rabbit anti-BAF [previously used (Kim et al., 2015 (link))].