Anti chk2

Cells were lysed in RIPA buffer containing Protease Inhibitor Cocktail (Roche). Sample proteins (20 μg) were separated by SDS-PAGE and then transferred to PVDF membranes. After blocking in 10% nonfat milk for 1 h at room temperature, membranes were incubated with primary antibodies overnight at 4°C or 2 h at room temperature. The membranes were then incubated with horseradish peroxidase-labeled secondary antibodies and visualized with ECL. The following primary antibodies were used: anti-p53 (1:500, CST), anti-phosphorylated p53 (Ser15) (1:500, CST), anti-acetylated p53 (1:800, CST), anti-Chk2 (1:500, BD Biosciences), anti-p21 (1:500, BD Biosciences), anti-p27 (1:1800, BD Biosciences), anti-p16 (1:1,200, SAB), anti-p19 (1:600, Thermo), anti-Rb (1:500, BD Biosciences), anti-p130 (1:800, Abcam), anti-Caspase 3 (1:800, CST), anti-PARP (1:800, CST), anti-Bax (1:500, CST), anti-CDK4 (1:800, CST), anti-CDK6 (1:800, CST), anti-E2F1 (1:500, BD Biosciences), anti-Foxo1 (1:500, Millipore), anti-Foxo3a (1:500, Millipore), anti-Sirt1 (1:800, CST), and anti-γ-tubulin (1:8000, Millipore).

Anti-BRCA1 [46 (link)] and anti-53BP1 [47 (link)] were gifts from Xiaochun Yu (City of Hope), anti-BARD1 [48 (link)] was a gift from Richard Baer (Columbia University). The follow antibodies were purchased: anti-γH2AX (Abcam, ab81299), anti-phospho-KAP1 S824 (Abcam, ab70369), anti-phospho-RPA2 (S4/S8) (Bethyl, A300-254A), anti-RAD51 (Santa Cruz, sc-8349), anti-CHK1 (Santa Cruz, sc-8408), anti-phospho-CHK1 S345 (CST, #2348S), anti-phospho-Histone H3 S10 (CST, #9701S), anti-RPA2 (CST, #2208S), anti-KAP1 (Sangon Biotech, D155285), anti-BrdU (BD biosciences, 347580), anti-CHK2 (BD biosciences, 611571), FITC-anti-mouse CD4 (BD biosciences, 557307), PE-anti-mouse CD8a (BD biosciences, 553032), and PE-anti-mouse TCRβ chain (BD biosciences, 561081).

The expression markers on T cells were determined by flow cytometry analyses after surface staining or intracellular staining with anti-human specific antibodies conjugated with PE or Alexa Flour488. These human antibodies included: anti-CD27 (clone: M-T271), anti-CD28 (clone: CD28.2), anti-2B4 (clone: 2–69), anti-PD-1 (clone: EH12.1), anti-CD57 (clone: HCD57), anti-KLRG1 (clone: 14C2A07), anti-CD160 (clone: BY55), anti-Tim-3 (clone: F38-2E2), anti-ATM (#2873), anti-H2AX (#7631), anti-CHK2 (#3440), anti-53BP (#4937), anti-AMPK (#2532), and anti-phospho-ATM (Ser1981, #5883), anti-phospho-H2AX (Ser139/Tyr142, #5438), anti-phospho-CHK2 (Thr68, #2661), anti-phospho-53BP (Ser25/29, #2674), and anti-phospho-AMPK (Thr172, #2535), which were purchased from BD Biosciences, Biolegend, or Cell Signaling Technology. The dilution of these antibodies was 1:100 for the studies. The cell gating strategy is shown in the Supplementary Fig. 8. All stained cells were analyzed on a FACS Calibur flow cytometer (BD Bioscience) and data analyzed with FlowJo software (Tree Star).