Dylight anti mouse and anti rabbit antibodies

Cells were collected by trypsinization and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma) with the addition of protease inhibitors (Roche) and phosphatase inhibitors (Sigma) for 30 minutes on ice. Insoluble material was removed by centrifugation at 14,000 rpm for 15 minutes at 4°C. Proteins were separated in 4% to 20% Tris-glycine polyacrylamide gels (Mini-PROTEAN; Bio-Rad) and electrophoretically transferred onto polyvinylidene fluoride membranes. Membranes were blocked with 3% BSA in PBS and incubated with antibodies against ϒH2AX and pATM (Abcam), p53 and p21 (Santa Cruz), pp53 Ser15 (Cell Signaling) and tubulin (Sigma). After incubation with primary antibodies, membranes were washed, incubated with secondary DyLight anti-mouse and anti-rabbit antibodies (Thermo Scientific), and signals was visualized using a Bio-Rad imager.

Immunoblotting was performed as previously described [25 (link),26 (link),27 (link)]. Briefly, cells were collected by trypsinization and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma) with the addition of protease inhibitors (Roche, Indianapolis, IN, USA) and phosphatase inhibitors (Sigma) for 15 min on ice. Insoluble material was removed by centrifugation at 14,000 rpm for 15 min at 4 °C. Proteins were separated in Tris-glycine polyacrylamide gels (Mini-PROTEAN; Bio-Rad, Hercules, CA, USA) and electrophoretically transferred onto polyvinylidene fluoride membranes. Membranes were blocked with 3% BSA in PBS and incubated with antibodies against ɣH2AX (Abcam, Cambridge, MA, USA), and tubulin (Santa Cruz, Dallas, TX, USA). After incubation with primary antibodies, membranes were washed, incubated with secondary DyLight antimouse and antirabbit antibodies (Thermo Scientific), and signals were visualized using a Bio-Rad imager. The uncropped immunoblotting image was shown in Figure S6.