Anti brg1 antibody

Cells were lysed in buffer containing 3% SDS, 30% glycerol, 150 mM Tris–HCl (pH7) and 5% β-mercaptoethanol. Protein extracts (30 μg) were loaded to a 10% SDS-PAGE gel and transferred to polyvinylidenefluoride (PVDF) membrane. Afterwards, the PVDF membranes were blocked in 5% milk for 2 h at room temperature and incubated with anti-Zscan4c antibody (1:5000 dilution, AB4340, Merck), β-Actin (1:5000 dilution, KM9001, Sungenebiotech), anti-Flag antibody (1:5000 dilution, 30503ES60, Yeasen), anti-HA antibody (1:5000 dilution, 30701ES60, Yeasen), anti-Brg1 antibody (1:2500 dilution, #49360, Cell Signalling Technology) or anti-Brd9 antibody (1:2500 dilution, #71232, Cell Signalling Technology) overnight at 4°C and subsequently incubated with goat anti-rabbit IgG-HRP (1:1000 dilution, sc-2004, Santa Cruz) or anti-mouse m-IgGκ BP-HRP (1:1000 dilution, sc-516102, Santa Cruz) for 90 min at room temperature. Membranes were treated with Immobilon Western HRP substrate (WBKLS0100, Merck) and images were taken by Chemiluminescence Imaging System (Tanon).

ChIP was done according to a published protocol (30 (link)). Approximately, 2 × 10⁷ ESCs were harvested for ChIP. Briefly, ESCs were cross-linked with 1% (w/v) formaldehyde for 10 min at room temperature and quenched in 200  mM glycine. Cells were washed with chilled tris-buffered saline (TBS) containing EDTA, scraped off the plates and collected by centrifugation. The collected cell pellet was lysed in buffer containing 0.25% Triton X-100 and protease inhibitors. Subsequently, chromatin pellet was collected and sonicated. Sample was pre-cleared with protein G beads at 4°C for 2 h and immunoprecipitated with anti-Flag antibody (M20008-M, Abmart), anti-H3K4me1 antibody (ab176877, Abcam), anti-H3K27ac antibody (ab177178, Abcam), anti-H3K14ac antibody (ab52946, Abcam), anti-Brg1 antibody (#49360, Cell Signalling Technology), anti-Brd9 antibody (#71232, Cell Signalling Technology) at 4°C overnight. Immunoprecipitated chromatins were subsequently eluted and decrosslinked. The immunoprecipitated DNA was purified with phenol:chloroform and analysed by qPCR.