Smooth muscle myosin heavy chain smmhc

Cell extracts were obtained by lysing VSMCs in complete Lysis-M buffer (Roche Diagnostics Corporation, Basel, Switzerland). Detergent-soluble fractions were retained, and protein concentrations in samples were determined using a Bradford protein assay (Bio-Rad, Hercule, USA). Lysates containing 30 μg of protein were electrophoresed on polyacrylamide gels (8% gel), transferred to Hybond-C nitrocellulose membranes (transblot turbo, Bio-Rad, Hercule, USA) and blotted with the following antibodies: α-smooth muscle actin (αSMA), 4/1,000 (Sigma-Aldrich), smooth muscle myosin heavy chain (SM-MHC), 1/1,000 (Abcam; Cambridge, UK); smoothelin, 1/500 (Santa Cruz Biotechnology, USA); integrin α_v, 1/1,000 (Santa Cruz Biotechnology, Dallas Texas); integrin β₃ 1/500 (Merck Millipore, Billerica, USA) and tubulin, 2/1,000 (Sigma-Aldrich). After rinsing, incubation with a secondary rabbit antibody 1/1,000 (α_v, β₃, smoothelin, SM-MHC, Sigma-Aldrich) and mouse antibody 1/1000 (αSMA, tubulin, Sigma-Aldrich). Reactions were visualized by the ECL Western Blot Detection Kit (Bio-Rad, Hercule, USA) after incubation with peroxidase conjugates 1/2000 (GE Healthcare, Little Chalfont, UK). tubulin was used as loading control and the protein expression was normalized to tubulin.