Chromatin Immunoprecipitation (ChIP) Assay

ChIP assays were performed essentially as described before^{22 ,26 (link),33 –44} . 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, 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, ab13370), anti-SRF (Cell Signaling Technology, 5147), (Santa Cruz, sc-585), anti-SLUG (Cell Signaling Technology, 9585), anti-ZEB1 (Cell Signaling Technology, 3396), anti-SNAIL (Cell Signaling Technology, 3879), anti-acetyl H3 (Millipore, 06-599), anti-acetyl H4 (Millipore, 06-598), anti-histone H3 (Millipore, 06-755), or pre-immune IgG. For re-ChIP, immune complexes were eluted with the elution buffer (1% SDS, 100 mM NaCO₃), diluted with the re-ChIP buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris pH 8.1), and subject to immunoprecipitation with a second antibody of interest.