Ab47433

Western blotting and immunohistochemistry were performed according to standard procedures^{50 (link)}. The following antibodies were used: anti-HBX (ab39716, Abcam, Cambridge, MA, USA), anti-P-CHK2 (2197S, Cell Signaling Technology, Danvers, MA, USA), anti-CHK2 (ab47433, Abcam), anti-ATM (ab199726, Abcam), anti-P21 (ab7960, Abcam), anti-P53 (BS1913, Bioworld, Minnesota, USA), anti-γH2AX (ab26350, Abcam), anti-P-CHK1 (2348S, Cell Signaling Technology), anti-CHK1 (10362-1-AP, Proteintech), anti-CDK2 (SC-6248, Santa Cruz Biotechnology, CA, USA), anti-Cyclin D1 (SC-718, Santa Cruz Biotechnology), anti-GAPDH (SC-47724, Santa Cruz Biotechnology), HRP-Ms (#7074, Cell Signaling Technology), and HRP-Rb (#7076, Cell Signaling Technology).

Total protein extracts were obtained from sub-confluent cell cultures of the cell lines described above. Cell cultures were incubated with 500 µL of cold lysis buffer (150 mM NaCl, 50 mM Tris-HCl [pH 7.5], 0,5% NP-40) in the presence of protease inhibitors (CompleteTM, Roche Diagnostics, Basel, Switzerland) and phosphatase inhibitors (PhosSTOP, Sigma-Aldrich). Extracts were cleared from debris by centrifugation (10,000× g for 20 min) at 4 °C, transferred to fresh tubes and sored at −80 °C until use. Protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Thirty μg of proteins were resolved by electrophoresis through sodium dodecyl sulfate (10 to 12%) polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The membranes were blocked for 1 h in 5% nonfat milk, probed with primary antibodies against ATM (Ab78), ATM (phospho S1981) (Ab81292), CHK2 (Ab47433), γH2AX (ab22551) from Abcam (Cambridge, MA, UK); and tubulin (sc-25259) from Santa Cruz Biotechnology (Santa Cruz, CA, USA), according to the manufacturer’s instructions. Membranes were reprobed with horseradish peroxidase (HRP)-conjugated secondary antibodies and revealed using enhanced chemiluminescence procedures according to the manufacturer’s recommendations (Amersham Pharmacia Biotech).