Western Blot Analysis of DNA Damage Response

Western blotting was performed as described previously [16 (link)]. Briefly, cells or tumors were lysed with RIPA buffer supplemented with protease inhibitor (#25,765,800, Sigma) and phosphatase inhibitor (#P5726, Sigma) for 30 min. 30 μg of lysates were separated by SDS-PAGE and transferred to nitrocellulose membrane, blocked with 5% BSA for 1 h at room temperature, then incubated with primary antibody overnight at 4 °C. Primary antibodies include: BRCA2 (#ab27976, Abcam); γ-H2AX ^S139 (#05–636, Millipore, 1:1000); H2AX (#2595, Cell Signaling, 1:1000); α-Tubulin (#2144, Cell Signaling, 1:1000); Actin (#A5316, Sigma 1:30,000); p-Chk1^S345(#2348, Cell Signaling, 1:1000); Chk1 (#2360, Cell Signaling, 1:1000); p-p53^Ser15(#82,530, Cell Signaling, 1:1000); p53(#2524, Cell Signaling, 1:1000); p-ATM^Ser1981(#4526, Cell Signaling, 1:1000); ATM (#2873, Cell Signaling, 1:1000); PD-L1 (#ab213480, Abcam, 1:1000). The next day, membranes were washed with PBST (0.1% Tween-20) three times, incubated with secondary antibody for 2 h at room temperature before developing with ECL substrate (#32,106, Thermo Fisher).