Anti trimethyl h3k4

Chromatin immunoprecipitation (ChIP) assays were performed essentially as described before. 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 (Brookfield, CT, United States). Aliquots of lysates containing 200 μg of protein were used for each immunoprecipitation reaction with anti-MRTF-A (Santa Cruz, sc-10768), anti-SRF (Cell Signaling Tech, 5147, Danvers, MA, United States), anti-BRG1 (Abcam, ab110641, Cambridge, United Kingdom), anti-acetyl histone H3 (Millipore, 06-599, Burlington, MA, United States), and anti-trimethyl H3K4 (Millipore, 07-442, Burlington, MA, United States). Precipitated genomic DNA was amplified by real-time PCR with the following primers: ET1 proximal promoter, 5′-GGCGTCTGCCTCTGAAGT-3′ and 5′-GGGTAAACAGCTCCGACTT-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,c (link)). All experiments were performed in triplicate wells and repeated three times.

Chromatin Immunoprecipitation (ChIP) assays were performed essentially as described before (Li et al., 2018d (link); Li Z. et al., 2019 (link); Shao et al., 2019 (link); Weng et al., 2019 (link); Yang et al., 2019 (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, 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-p300 (Santa Cruz, sc-585), anti-c-Jun (Santa Cruz, sc-1694), anti-Fos (Santa Cruz, sc-166940), anti-SMAD3 (Abcam, ab28379), anti-ASH2 (Bethyl Laboratories, A300-489A), anti-JMJD2B (Bethyl Laboratories, A301-478), anti-anti-acetyl H3 (Millipore, 06-599), anti-trimethyl H3K4 (Millipore, 07-449), anti-trimethyl H3K9 (Millipore, 07-441), 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.