Immunoprecipitation assay buffer

Cells were lysed at 4°C in ice-cold immunoprecipitation assay buffer (Cell Signaling Technology, 9806) and cell lysates were cleared by brief centrifugation (13,000 g, 15 min). Concentrations of proteins in the supernatant were determined using the BCA assay. For immunoprecipitation assay, cell lysate was incubated with anti-Flag M2 beads or IP-proved antibodies overnight at 4 °C on a rotator, followed by the addition of protein A/G plus agarose to the reaction for 2 h at 4 °C. To detect ubiquitin conjugates, the lysates were denatured in lysis buffer containing 1% SDS to disrupt any protein-protein interactions, and then diluted ~10 fold prior to IP followed by immunoblot. Prior to immunoprecipitation, samples containing equal amounts of proteins were pre-cleared with protein A agarose beads (Santa Cruz Biotechnology, sc-2027; 4°C, 3 h), and subsequently incubated with various irrelevant IgG or specific antibodies in the presence of protein A agarose beads for 2 h or overnight at 4°C with gentle shaking. After five washes with lysis buffer supplemented with protease inhibitor mixture, complexes were released from the anti-Flag M2 beads by boiling for 5 min in 2x SDS-PAGE loading buffer.

MCF-7 breast cancer cells were treated with HANP/GKT831 (0.5 and 1 μM of equivalent dose of GKT831) for 24 h and then received 5 Gy of irradiation. Cells were collected 4 h after irradiation and lysed in an immunoprecipitation assay buffer (Cell Signaling Technology). Protein extracts of cells were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then blotted onto poly(vinylidene difluoride) membranes. Blots were incubated with rabbit antihuman γH₂AX antibody (sc-10790, Santa Cruz Biotechnology) and then horseradish peroxidase (HRP) conjugated goat antirabbit IgG antibody (Santa Cruz Biotechnology). Positive protein bands were detected using the G:BOX Systems Imaging System (Syngene).