Anti rabbit igg a6667

General tissue culture materials were obtained from VWR International. Antibodies against BRD2 (ab139690), BRD3 (ab50818), BRD4 (ab128874) and IRF1 (ab26109) antibody for chromatin immunoprecipitation (ChIP) were obtained from Abcam. Antibodies against c-MYC (5605), human PD-L1 (E1L3N clone; 13684), IRF1 (8478) and control IgG (2729) antibody for ChIP were purchased from Cell Signaling Technology. Anti-BRD4 (A301–985A) antibody for ChIP was obtained from Bethyl Laboratories. PE-conjugated human PD-L1 (MIH1 clone; 12-5983-42) antibody was from Thermo Fisher. The GAPDH (MAB374) antibody was from Millipore, and α-tubulin (sc-8035) antibody and HSP90 (sc-7940) antibody were obtained from Santa Cruz Biotechnology. Secondary anti-mouse IgG (A4416) and anti-rabbit IgG (A6667) antibodies were purchased from Sigma. The BET inhibitors JQ1 and I-BET151 were obtained from Tocris Bioscience, the BET PROTAC ARV-825 was obtained from MedChem Express, and human and mouse IFN-γ was purchased from Thermo Fisher Scientific. The siRNAs against BRD2, BRD3, BRD4, c-MYC, and IRF1 were purchased from Thermo Fisher Scientific.

Whole cell extracts of cultured cells were prepared in radioimmunoprecipitation (RIPA) lysis buffer supplemented with phosphatase and protease inhibitors (Calbiochem, Burlington, MA, USA). Protein concentration was determined by the bicinchoninic acid assay (BCA) (Thermo Fisher Scientific, Waltham, MA, USA) and separated by SDS-PAGE gel. The following antibodies and dilutions were used: hnRNPA1 (1:2000, Cell Signaling, Danvers, MA, USA), cleaved PARP1 (1:1000, Cell Signaling), HSP90 (1:4000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), GAPDH (1:3000, EMD Millipore, Billerica, MA, USA), Survivin (1:2000, Cell Signaling), and Flag-tag (1:2000, Sigma-Aldrich). Following blocking with 5% BSA, membranes were incubated with the primary antibodies overnight at 4 °C. The next day, membrane was incubated with secondary antibodies for 1 h at room temperature. Secondary anti-mouse IgG (A4416) and anti-rabbit IgG (A6667) antibodies were purchased from Sigma-Aldrich and used at a 1:4000 dilution. Images of blots were acquired on HyBlot ES Autoradiography Film (Thomas Scientific, Swedesboro, NJ, USA) following incubation with SuperSignal West Pico PLUS (Thermo Fisher Scientific). When necessary, membranes were stripped using Restore Western Blot Stripping Buffer (Thermo Fisher Scientific) according to manufacturer’s instructions.