Sc 21558

Cells were lysed in extraction buffer (25 mM Tris, pH 7.4, 50 mM sodium chloride, 0.5% sodium deoxycholate, 2% NP-40, 0.2% SDS) containing protease inhibitors. Total cell lysates were separated on 4–20% SDS-PAGE (Invitrogen), transferred to nitrocellulose membranes (Millipore), immunoblotted with antibodies, and visualized using a SuperSignal West Dura Extended Duration Substrate (Thermo Scientific). The antibodies used in this study are anti-SMA (Sigma, A2547, 1:4000 dilution), anti-type I collagen (Rockland, 600-401-103, 1:1000 dilution), anti-MRTFA (Santa Cruz, sc-21558, 1:250 dilution), anti-ACLP (1:4000 dilution)^{27 (link)} and anti-GAPDH Sigma, G9545, 1:10,000 dilution). Western blots were quantitated using ImageJ and normalized to GAPDH. The results are expressed as fold change versus control.

ChIP was performed as previously described (75 (link)) with slight modifications. Briefly, chromatin was cross-linked with 1% formaldehyde, cells were incubated in lysis buffer (1% SDS, 0.01 M EDTA, 0.05 M Tris pH 8.1, 0.1 mM PMSF in isopropanol) and DNA was fragmented into approximately 500-bp fragments using a Bioruptor (Diagenode Pico Bioruptor). Aliquots of lysates containing 13 μg chromatin were used for immunoprecipitation using anti-MRTFA (Santa Cruz Biotechnology, sc-21558) or goat IgG (76 (link)). Cross-links were reversed by heating at 65°C in the presence of 0.2 M NaCl and DNA fragments were recovered using the ChIP DNA Clean and Concentration Kit from Zymo Research (catalog D5205). Primers for the promoter region (Supplemental Table 2) were located within 400 bp upstream of the transcription start site (52 (link)).