P65 pser536

Whole-cell lysate was generated with RIPA lysis and extraction buffer supplemented with protease inhibitors and phosphatase inhibitors (Thermo, Canoga Park, CA, USA); fractionated cell lysates were generated with NE-PER nuclear and cytoplasmic extraction reagents (Thermo). Electrophoresis and transfer were based on standard procedures as previously reported.^{47 (link)} Protein blots were probed with primary antibodies against Actin (Cell Signaling Technology #4970), Histone3 (Cell Signaling Technology #4499), RAP1 (Cell Signaling Technology #5433), p65 (Cell Signaling Technology #8242), p65-pSer536 (Cell Signaling Technology #3033), IκBα (Cell Signaling Technology #4814), IκBα-pSer32 (Cell Signaling Technology #2859), γH2AX (Abcam, Cambridge, UK #11174), BCL-2 (Cell Signaling Technology #2870) and cleaved caspase-3 (Cell Signaling Technology #1658) followed by HRP-conjugated secondary antibodies (Cell Signaling Technology #7074 and #7076). Immunoblots were detected using ECL reagents (Thermo).

Whole cell lysates of lung cancer samples were prepared with a proteinase and phosphatase inhibitor cocktail (Roche, CA, USA). Protein concentrations of lysates were measured using the BCA method (Thermo Fisher Scientific, USA). 20 µg of extracted protein was loaded on to a 10% SDS-polyacrylamide gels and then transferred onto PVDF membranes (Millipore, USA). Non-specific binding was blocked on the PVDF membranes with PBS buffer containing 0.1% Tween-20, 2% BSA, and 5% nonfat dry milk. Blots were then incubated with anti-rabbit primary antibodies overnight at 4 °C. The next day, membranes were extensively washed, and then incubated with horseradish peroxidase-conjugated anti-goat (Proteintech) or anti-rabbit (Proteintech) IgG at room temperature for 1 h. Protein blots were probed with primary antibodies against CHD1L (Abcam#ab197019), p65 (Cell Signaling Technology #8242), p65-pSer536 (Cell Signaling Technology #3033), IκBα (Cell Signaling Technology#4814), and IκBα-pSer32 (Cell Signaling Technology #2859). Signals were visualized by chemiluminescence (Bio-rad, Hercules, California) and quantitated using a Quantity One system (Bio-Rad, Hercules, CA, USA).