D4k9n

Total protein was extracted using a Total Protein Extraction Kit (Sangon, China), and protein concentration was measured with a BCA Protein Assay Kit (Beyotime, China). Protein samples were separated by 10% SDS polyacrylamide gels and electrotransferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). PVDF membranes were blocked and then incubated with polyclonal antibodies against ASIC3 (Alomone Labs, Israel, ASC-018), α-SMA (CST, USA, D4K9N), Collagen I (Abcam, USA, ab138492), CD206 (Novus Biologicals, USA, MAB2534), PI3K (Abcam, USA, ab191606), p-PI3K (Abcam, USA, ab182651), AKT (Abcam, USA, ab179463), p-AKT (Abcam, USA, ab38449), or GAPDH (Abcam, USA, ab8245) overnight at 4 °C and then incubated with horseradish peroxidase-conjugated secondary antibody (Abcam, USA, 7074) at room temperature for 1 h. GAPDH served as an internal control for protein normalization.

The aorta tissue was homogenized and centrifuged at 13,000 rpm for 30 min at 4 °C to take the supernatant. The extract of aorta tissues was prepared in lysis buffer [20 mM Tris-HCl, 1 mM dithiothreitol (DTT), 5 mM EGTA, 0.5 mM PMSF, 20 μM leupeptin, and 20 μM aprotonin]. Separation of total protein were done by using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 10–14% acrylamide gels, then transferred to a polyvinylidine diflouride (PVDF). After blocking with 5% fat-free milk for 1 hour, the membranes were incubated overnight at 4 °C with the respective primary antibodies rabbit antibodies against SM22α (1:1000, ab14106 Abcam, Cambridge, MA, USA), α-SMA (1:1000, D4K9N, USA), BMP-2 (1:500, ab14933, Abcam, Cambridge, MA, USA), Runx2 (1:100, sc-390351, Santa Cruz, Dallas, TX, USA), Nrf2 (1:500, tcba 1560, Taiwan), Keap1 (1:1000, 10503-2-AP, USA), and HO-1 (1:250, sc-390991, Santa Cruz, Dallas, TX, USA). The blots were visualized with enhanced chemiluminescence (ECL) system (Millipore Corporation, Billerica, MA 01821 USA) and Image J software was used to scan and quantify the gray values.