Anti γ h2ax

The primary antibody, anti-CAMR1 (1:1,000; sc5418), was purchased from Upstate Biotechnology (Lake Placid, NY, USA). Anti-Bax (1:1,000; clone 6A7), anti-Bcl-2 (1:1,000; Cat sc-509), and anti-γ-H2AX (1:1,000; Ser139) were acquired from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-β-actin (1:10,000; A1978) was obtained from Sigma-Aldrich (Louis, MO, USA). All these antibodies are monoclonal antibodies. Cells were harvested and total proteins were isolated with RIPA reagents (Thermo Scientific, Rockford, IL, USA), according to the manufacturer’s instruction. Subsequently, protein concentrations were detected using the BCA kit (Pierce, Rockford, IL, USA). Western blot analysis was performed following previous protocols.20 (link) Briefly, protein specimens were isolated by a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). After blocking in Tris Buffered saline with Tween (TBST) buffer with 5% BSA for 2 h at 37°C, the membranes were incubated with primary antibodies overnight at 4°C. Following washing in TBST, the membranes were further incubated with secondary antibody labeled with horseradish peroxidase for 1 h at room temperature. The proteins were visualized using the Image Quant software (Molecular Dynamics, Sunnyvale, CA, USA).

Total proteins were extracted using the ProteoPrep Total Extraction Sample Kit (Sigma-Aldrich, USA). Western blot was performed using the primary antibodies, followed by horseradish peroxidase-conjugated secondary antibodies and detection by an enhanced chemiluminescence reagent (Pierce, USA). The primary antibodies used were anti-γH2AX, anti-Nrf2, anti-NQO-1, anti-HO-1, and anti-GAPDH from Santa Cruz Biotechnology (Santa Cruz, USA), anti-caspase-3 and anti-Chk1 from Cell Signaling Technology (CST, USA), and anti-caspase-8 and anti-caspase-9 from Becton Dickson (BD, USA). Secondary antibodies were from Cell Signaling Technology (CST, USA).

IHC was performed as described previously (18 (link)). Briefly, after deparaffinization and blocking, sections were incubated with primary antibodies at 4°C overnight. After washing with PBS, the sections were incubated with a secondary antibody for 1 h at RT. Then, 3,3′-diaminobenzidine (DAB) substrate was used to develop color, and hematoxylin was used for counterstaining. The primary antibodies were anti-APE1 (mouse monoclonal, cat. no. 17774, dilution 1:500; Santa Cruz Biotechnology, Inc.) and anti-γ-H2AX (rabbit monoclonal, cat. no. 9718, dilution 1:50; Cell Signaling Technology, Inc.).