Anti rad52

The SW1736 and BHP7-13 cells treated with 1.5, 6.0, and 24.0 μM of JR-P(II) for 48 h were collected and then lysed using lysis buffer. The buffer contained Tris-HCl (40 mM; pH 7.4), sodium chloride (150 mM), and Triton X-100 (1% v/v), along with the protease inhibitors. Lysate centrifugation for 20 min at 12 000 g at 4°C was followed by determination of protein concentration in supernatant using a bicinchoninic acid protein kit. The protein samples (30 μg per lane) were subjected to resolution by electrophoresis on 10% SDS-PAGE followed by transfer to PVDF membranes. The membrane blocking on incubation with 5% skimmed milk in TBS plus Tween-20 (0.1%) was carried out for 2 h at room temperature. The samples were subjected to probing on incubation with primary antibodies anti-caspase-3, anti-p-AKT, anti-p-ERK1/2, anti-p-S6, anti-p-H2AX, anti-KU70, anti-KU80, anti-p-4E-BP1, anti-RAD52, anti-ERCC1 anti-PCNA, and α-tubulin primary antibodies (Cell Signaling) overnight at 4°C. Then, 1X PBST washing of membranes was followed by incubation for 2 h with horseradish peroxidase-conjugated secondary antibody. The immunoblots were visualized using SignalFire™ Plus ECL Reagent and quantified using Image J version 2.0 software.