Mouse monoclonal anti phospho γh2ax

MDA-MB468 cells were lysed with TNN buffer^{7 (link)}. An aliquot of cell lysates was lysed with SDS lysis buffer and the rest of cell lysates were incubated with appropriate antibodies or beads at 4° C for 3-12 h. Anti-FLAG beads (Sigma) were washed three times with TNN buffer. Immunoprecipitates were fractionated by SDS-PAGE and electro-transferred to Immobilon-P membrane (Millipore). Equal amount of protein loading was validated with Ponceau-S staining. The specific signals were detected by incubating with appropriate antibody. All primary antibodies were used at 1:1000 dilution and horseradish peroxidase (HRP)-conjugated secondary antibodies were used at 1:5000 dilution for immunoblotting. E2F1 (C-20 and KH-95), p53 (FL393), p63 (4A4 and H-129), p73 (H-79), p21 (C-19), Bax (N-20), GST (B-14), c-Myc (A14), Miz1 (H-190), Chk1 (G4), Actin (C-2) and GAPDH (6C5) antibodies were purchased from Santa Cruz. TopBP1 (mouse monoclonal) and Akt antibodies were purchased from BD Transduction Laboratories. TopBP1 (BL893, Rabbit polyclonal) antibody was purchased from Bethyl. Mouse monoclonal anti-phospho-γH2AX was purchased from Millipore. Phospho-Chk1 (Ser345) antibody was purchased from Cell signaling. FLAG (F7425) antibody was purchased from Sigma. Mouse monoclonal anti-p73 (IMG-246) was purchased from IMGENEX. Full blots are shown in Supplementary Fig. 19.