Anti myosin

Detailed experimental methods for Western Blot analysis are described in-depth by Han et al. (Han et al., 2019a). Antibodies used for experiments included: anti-Myosin (Santa Cruz Biotechnology, CA, USA; 1:200 dilution), anti-MyoD (Santa Cruz Biotechnology; 1:500 dilution), anti-caspase-3 (Santa Cruz Biotechnology; 1:1000 dilution), anti-caspase-9 (Santa Cruz Biotechnology; 1:1,000 dilution), anti-AKT, anti-p-AKT (Santa Cruz Biotechnology; 1: 1000 dilution), anti-IGF2BP3, anti-IGF2 (abclonal, Wuhan, China; 1:1000 dilution), and β-actin (ZENBIO, Beijing, China, 1: 5000 dilution). β-actin was used as a loading control.

SMSCs were seeded in 6-well plates and cultured in DM. After transfection for 72 h, cells were collected and proteins were extracted using lysis buffer and the concentration determined by a bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China). Total proteins (30 μg) were separated on a 12% SDS-PAGE, and transferred to a 0.2 mm polyvinylidene fluoride (PVDF) membrane that was soaked in formaldehyde and then blocked with 5% skim milk in Tris saline with Tween (TBST) buffer for about 2h at room temperature. The membrane was then incubated overnight with primary antibodies specific for anti-Myosin (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:500), anti-MYOG (Santa Cruz Biotechnology; 1:500), and anti-β-actin (Santa Cruz Biotechnology; 1:1000) at 4 ℃. The PVDF membrane was washed three times with TBST buffer and then incubated with secondary antibody for 2 h at room temperature. β-actin was used as the internal control with a secondary antibody that was horseradish peroxidase (HRP)-labeled anti-mouse immunoglobulin G (IgG) (ZenBio, Beijing, China; 1:2000). Finally, antibody reacting bands were detected using enhanced chemiluminescence (ECL) luminous fluid (Solarbio, Beijing, China).