Ab15734

Total proteins from the isolated cells and tissues were extracted in TNN buffer (50 mM Tris-Cl, pH 7.4, 1% NP-40, 150 mM NaCl, and 1 mM EDTA) supplemented with protease inhibitors (1 mM PMSF, 1 μg/mL aprotinin and 1 μg/mL leupeptin) and phosphatase inhibitors (1 mM Na₃VO₄ and 1 mM NaF). Protein samples (15 μg) were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Bio-Rad, Hercules, California, CA, USA). After blocking non-specific antibody binding sites, the membrane was incubated overnight at 4 °C with a mouse monoclonal antibody against α-SMA (ab15734, Abcam, Cambridge, MA, USA), collagen I (ab138492, Abcam), E-cadherin (sc-8426, Santa Cruz Biotechnology, Paso Robles, CA, USA), fibronectin (sc-8422, Santa Cruz Biotechnology), N-cadherin (sc-393933, Santa Cruz Biotechnology), MMP2 (13132, Cell signaling, Danvers, MA, USA), vimentin (sc-32322, Santa Cruz Biotechnology) and β-actin (A5316, Sigma–Aldrich). All the antibodies were used at a dilution of 1:1,000. After incubation with peroxidase-conjugated secondary antibodies at 37 °C for 1 h, the generated protein bands were visualized using an enhanced chemiluminescence reagent (Cyanagen, Bologna, Italy) and detected using the Amersham Imager 680 (GE Healthcare Biosciences, Little Chalfont, UK). All experiments were performed in triplicate.

Protein from HASMCs subjected to stretch or not was extracted by use of a cytoplasmic extraction reagent kit (USA). The protein concentrations were determined by the BCA method. Western blot analysis was performed to detect the protein level. In brief, 15–20 µg protein extract was separated by SDS-PAGE and transferred to PVDF membrane (USA), which was blocked with 5% nonfat milk for 2 hr. Then membranes were incubated with primary antibodies overnight at 4°C. After a washing with 1×TBST 3 times, membranes were incubated with secondary antibodies for 1 hr. Visualization involved an enhanced chemiluminescence-plus detection system. Relative band intensities were analyzed by use of Photoshop CS3. The major primary antibodies used were as follow: anti-ACE antibody (abcam, ab28611), anti-CNN antibody (epitomics, #1806-1), anti-SMA(abcam, ab15734), anti-SM22α (abcam, ab14106), anti-SRF (Cell signaling technology, CST, #5147), anti-Kruppel-like factor (KLF4) (CST, #4038), anti-Myocardin (abcam, ab22073), total and phospho-MAPK antibody (CST, #9926, #9910).