Brd9 antibody

Cells were washed with ice-cold PBS and lysed using ice-cold lysis buffer (Hepes 20 mM pH 8, EDTA 0.2 mM, NaCl 0.3 mM, Nonidet P-40 0.5%, 15% glycerol) and mixture protease inhibitor (Sigma-Aldrich, 124469). Protein concentrations in the extracts were measured using Bradford and were made equal in different samples with the extraction reagent. Lysates were mixed with sample loading buffer and denatured at 95 °C for 10 min. Samples were separated by SDS/PAGE, transferred onto PVDF membrane. Immunoblot analysis was performed with BRD9 antibody (Active Motif, cat. no. 61537) and secondary anti-rabbit antibodies conjugated to horseradish peroxidase.

Cell pellets were resuspended in cytosolic lysis buffer (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.4% NP-40, Protease Inhibitor [1X] [Roche]) and shaken on ice for 15 min. After centrifugation at 3,000g for 3 min, pellets were washed once with cytosolic buffer and resuspended in nuclear lysis buffer (20 mM HEPES pH 7.9, 0.4M NaCl, 1 mM EDTA, 10% Glycerol, Protease Inhibitor [1X] [Roche]) and sonicated. Membranes were incubated with BRD9 antibody (Active Motif, Catalog: 61,537) at 1:1,000, BRD7 antibody (Cell Signaling, D9K2T) at 1:1,000, and HDAC1 antibody (Santa Cruz, sc-7872) at 1:500 overnight at 4°C.