Anti mkl1

Whole cell protein extraction and nuclear protein extraction were essentially performed as previously described (Shao et al., 2019 (link); Weng et al., 2019 (link); Yang et al., 2019a (link), b (link); Zhao et al., 2019 (link)). Specific antibodies or pre-immune IgGs (P.I.I.) were added to and incubated with cell lysates overnight before being absorbed by Protein A/G-plus Agarose beads (Santa Cruz). Precipitated immune complex was released by boiling with 1X SDS electrophoresis sample buffer. Western blot analyses were performed with commercially available antibodies: anti-RhoJ (Abcam, ab105311), anti-MKL1 (Santa Cruz, sc-32909), anti-ERG1 (Santa Cruz, sc-353), anti-FLAG (Sigma, F3165), anti-GFP (Proteintech, 50430-2), and anti-μ-actin (Sigma, A2228). Image J software was used for densitometrical quantification and densities of target proteins were normalized to those of μ-actin. Data are expressed as relative protein levels compared to the control group which is arbitrarily set as 1. All experiments were repeated at least three times.

Chromatin Immunoprecipitation (ChIP) assays were performed essentially as described before^{45 (link)–49 (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-MKL1 (Santa Cruz, sc-32909), anti-STAT3 (Cell Signaling Technology, 9132), 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.

Chromatin Immunoprecipitation (ChIP) assays were performed essentially as described before [45 (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 ∼500 bp pieces using a Branson 250 sonicator. Aliquots of lysates containing 200 μg of protein were used for each immunoprecipitation reaction anti-FLAG (Sigma, A2220, 1:100), anti-MKL1 (Santa Cruz, sc32909, 1:100), anti-p65 (Cell Signaling, 8242, 1:100), anti-HDAC5 (Santa Cruz, sc133225, 1:100) or pre-immune IgG. Precipitated genomic DNA was amplified by real-time PCR with the previously described primers.

ChIP assays were performed essentially as described before^{[20 (link)}–^{21 (link)]}. Aliquots of lysates containing 200 μg of nuclear protein were used for each immunoprecipitation reaction with anti-MKL1 (Santa Cruz), anti-BRG1 (Abcam), anti-WDR5 (Bethyl Laboratories), anti-dimethylated H3K4 (Millipore/Upstate), and anti-trimethylated H3K4 (Millipore/Upstate). For re-ChIP, immune complexes were eluted with the elution buffer (1% SDS, 100mmol/L NaCO₃), diluted with the re-ChIP buffer (1% Triton X-100, 2 mmol/L EDTA, 150 mmol/L NaCl, 20 mmol/L Tris pH 8.1), and subject to immunoprecipitation with a second antibody of interest. All experiments were repeated three times.

Whole cell lysates were obtained by re-suspending cell pellets in RIPA buffer (50 mM Tris pH7.4, 150 mM NaCl, 1% Triton X-100) with freshly added protease inhibitor (Roche). Specific antibodies or pre-immune IgGs (P.I.I.) were added to and incubated with cell lysate overnight before being absorbed by Protein A/G-plus Agarose beads. Precipitated immune complex was released by boiling with 1X SDS electrophoresis sample buffer. Alternatively, FLAG-conjugated beads (M2, Sigma, A2220) were added to and incubated with lysates overnight. Precipitated immune complex was eluted with 3X FLAG peptide (Sigma). Western blot analyses were performed with anti-FLAG (Sigma, F3165, 1:3,000), anti-HA (Sigma, H9658, 1:3,000), anti-β-actin (Sigma, A1978, 1:3,000), anti-MKL1, (Santa Cruz, sc32909, 1:1,000), anti-HDAC5 (Abcam, ab1439, 1:2,000), and anti-acetyl lysine (Cell Signaling Tech, 9441, 1:1,000) antibodies. All experiments were repeated at least three times.

For cells, lysates were obtained by re-suspending cell pellets in RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100) with freshly added protease inhibitor tablet (Roche). For tissues, lysates were obtained by homogenizing samples in lysis buffer (10 mM Tris pH 8.0, 130 mM NaCl, 1% Triton-X100). Western blot analyses were performed with anti-β-actin (Sigma; 1∶5,000), anti-collagen type I (Rockland; 1∶5,000), and anti-MKL1 (Santa Cruz; 1∶1,000) antibodies.

Immunohistochemistry was performed as previously described [20] (link). Briefly, the sections were blocked with 10% normal goat serum for 1 hour at room temperature and then incubated with anti-MKL1 (Santa Cruz; 1∶100), anti-sm-MHC (Sigma; 1∶100), anti-α-SMA (Sigma; 1∶100), anti-CD3 (BD Bioscience; 1∶100), anti-CD45 (Abcam; 1∶100), or anti-F4/80 (Abcam; 1∶100) antibodies. Staining was visualized by incubation with an appropriate biotinylated 2° antibody and developed with a streptavidin-horseradish peroxidase kit (Pierce) for 20 min. Sections were counterstained with hematoxylin. Pictures were taken using an Olympus IX-70 microscope. Picrosirius red, Masson's trichrome (both from Sigma), and elastica von Gieson (Millipore) stainings were performed according to vendor's recommendations.