Anti stat1

Nuclear extracts were prepared and analyzed by immunoblotting, as described (Paulson et al., 1999 (link)). In brief, cells were lysed in RSBG40, soluble nuclear proteins were extracted from pelleted nuclei in RSB supplemented with 150 mM NaCl, and chromatin-bound proteins were subsequently extracted in 400 mM NaCl buffer. Alternatively, whole-cell lysates were prepared as described (Paulson et al., 1999 (link)). Antibodies used were anti-IRF9 (Veals et al., 1993 (link)), anti-STAT1, anti-STAT2, anti-phospho-STAT1 and anti-phospho-STAT2 (Invitrogen), anti–α-tubulin (T9026; Sigma), anti-HDAC1 (clone 2E10, 05-614; Millipore Sigma), anti-HDAC2 (clone 3F3, 05-814; Millipore Sigma), anti-E2F4 (A-20, sc-1082; Santa Cruz Biotechnology), anti-Sin3B (AK-12; Santa Cruz Biotechnology), anti–NELF-E (H140; Santa Cruz Biotechnology), anti-Brd4 (A-301-985A100; Bethyl Laboratories), anti-actin (Clone C4, MAB1501; Millipore Sigma). Luciferase assays were performed using standard methods as previously described (Marié et al., 2000 (link)).

Whole-cell lysates were prepared on ice using RIPA lysis buffer solution (Thermo Fisher), 1% protease, and phosphatase inhibitor cocktail (Invitrogen). Protein concentrations were determined with the Protein BCA Assay (Thermo Fisher). Lysate protein was subjected to 10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. After blotting, membranes were probed with a primary antibody at 4°C overnight. The following primary antibodies were used: anti-SP140 (MyBioSource), anti-STAT5a (Invitrogen), anti-phospho-STAT5a^Y694 (Invitrogen), anti-STAT1 (Invitrogen), and anti-phospho-STAT1^Y701 (p-STAT1^Y701) (Cell Signaling Technology). After washing three times with Tris-buffered saline with 0.1% Tween 20 Detergent (TBST), membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary goat anti-rabbit (Bio-Rad) or anti-mouse (Bio-Rad) antibodies as indicated for 2 hours at room temperature. Blots were developed with the Clarity Max Western ECL detection system (Bio-Rad) according to the manufacturer’s instructions, and images were captured using an iBrightCL1000 (Invitrogen).