Sc 10768

Chromatin immunoprecipitation (ChIP) assays were performed essentially as described before (Li et al., 2019b , f , 2020a (link), b , c (link); Shao et al., 2019 (link); Weng et al., 2019 (link); Chen et al., 2020a ; Wu X. et al., 2020 (link); Hong et al., 2021 (link)). In brief, chromatin in control and treated cells were cross-linked with 1% formaldehyde. Cells were incubated in lysis buffer (150 mM NaCl, 25 mM Tris pH 7.5, 1% Triton X-100, 0.1% SDS, and 0.5% deoxycholate) supplemented with protease inhibitor tablet and PMSF. DNA was fragmented into ∼200 bp pieces using a Branson 250 sonicator. Aliquots of lysates containing 200 μg of protein were used for each immunoprecipitation reaction with anti-BRG1 (Santa Cruz, sc-10768), anti-Sp1 (Abcam, ab227383), or pre-immune IgG. Precipitated genomic DNA was amplified by real-time PCR with the following primers: SCAP proximal promoter, 5′-ATACTTCCCTCCGGTGTCCAC-3′ and 5′-ACCTCTCACCTCCACCTTTAC-3′; SCAP distal promoter, 5′-AAATGCGAGGACATGTACAATAC-3′ and 5′-ATTTAAAAGCTAAGTTGAC-3′. A total of 10% of the starting material is also included as the input. Data are then normalized to the input and expressed as % recovery relative the input as previously described (Chen et al., 2020b (link), c (link)). All experiments were performed in triplicate wells and repeated three times.

SMARCA4 (BRG1) and SMARCA2 (BRM) protein expression was assessed by immunohistochemistry (IHC) using the anti-BRG1 (Santa Cruz, sc-10768) and anti-BRM (Abcam, ab15597) antibodies at dilutions of 1/200 and 1/50, respectively. After paraffin removal and hydration, slides were immersed in 10 mM citrate buffer pH 6 for 30 min for antigen retrieval, incubated with primary antibody for one hour at room temperature, washed and incubated with biotinylated secondary antibody for 30 min at room temperature. Streptavidin-biotin amplification (VECTASTAIN Elite ABC Kit) was then performed for 30 min, followed by peroxidase/diaminobenzidine substrate chromogenic reaction. IHC for SOX2 was performed using a Bond III automated immunostainer and the Bond Polymer Refine detection system (Leica Microsystems, IL, USA). Slides were deparaffinized and heat-mediated antigen retrieval was performed using the Bond Epitope Retrieval 2 solution at pH 9 (H2). The anti-SOX2 antibody clone D6D9 (Cell Signaling Technology) was used at 1/100 dilution.