Anti srf

hES-MCs were cultured in αMEM or treated with TGF-β. Cells were washed two times with cold PBS, followed by protein extraction using radioimmunoprecipitation assay buffer (50 mmol/l Tris-HCI, pH 7.4, 1% Triton X-100, 0.25% [wt/vol] sodium deoxycholate, 150 mmol/l NaCl, 1 mmol/l ethylene glycol tetraacetic acid, 0.1% SDS, protease inhibitors, and phosphatase inhibitors). Protein concentration was measured using BCA Protein Assay reagent (Thermo Scientific, Waltham, MA ). A 5- or 10-μg amount of the lysates was resolved by SDS–PAGE and transferred to polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA). Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline and Tween 20 and then incubated with primary antibodies in the blocking buffer for 1–2 h, followed by incubation with horseradish peroxidase–conjugated secondary antibody for 1 h (Sigma-Aldrich, St. Louis MO). Signal detection was performed with enhanced chemiluminescence (Millipore, Billerica, MA). Antibodies used were anti-Olfm2 (Abcam, Cambridge, MA), anti-SRF (Santa Cruz Biotechnology, Dallas, TX), anti–α-SMA (Abcam), anti-SM22α (Abcam), anti-SMMHC (Biomedical Technologies, Stoughton, MA), anti–α-tubulin (Cell Signaling, Danvers, MA), anti-FLAG (Sigma-Aldrich), and anti-HERP1 (Santa Cruz Biotechnology).

Total protein samples were prepared from fresh-frozen tissues or cultured cells by complete cell lysis (Roche, Mannheim, Germany) with protease and phosphatase. Cytoplasmic and nuclear proteins were isolated using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Jiangsu, China). Approximately 20–50 μg of denatured protein was separated by SDS-PAGE and transferred to nitrocellulose filter membranes (Millipore, Billerica, MA, USA). The antibodies used are listed as follows: anti-SRF (Santa Cruz Biotechnology, Dallas, TX, USA), anti-E-cadherin (Santa Cruz Biotechnology), anti-vimentin (Santa Cruz Biotechnology), anti-histone H3 (Santa Cruz Biotechnology), anti-tubulin (Santa Cruz Biotechnology), and anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA). The bands were scanned using a ChemiDoc XRS+Imaging System (Bio-Rad, Hercules, CA, USA). The absorbance of the bands was analyzed using image analysis software (ImageJ 1.48) and values were expressed as percentages of SRF/Histone or SRF/Tubulin ratio.

Indirect immunofluorescence staining for SRF and β-catenin was performed on GC cells and frozen sections of xenografted tumor tissues using anti-SRF (Santa Cruz Biotechnology) and anti-β-catenin (Cell Signaling, Danvers, MA, USA) antibodies as previously described.^{43 (link)}