Ab108519

Proteins were extracted from the HEK293T transfected with the mixture of equivalent amount of miR-30-5p constructs, using RIPA buffer (Solarbio; Beijing, China) containing 1 mM PMSF (Solarbio; Beijing, China). Then, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed in the 8% or 12% polyacrylamide gels with 20 μg proteins per lane. After this, the proteins in the gels were transferred onto the PVDF membrane that then were blocked with 5% skim milk in Tris Buffered Saline with Tween (TBST) buffer (0.15 M NaCl, 1 M Tris–HCl pH 8.0, 0.05% Tween-20) for 2 h at room temperature. After incubation with primary antibody against MBNL1 (dilution 1:1000; ab108519; Abcam, Shanghai, China), MyoG (dilution 1:1000; ab124800; Abcam, Shanghai, China), MHC (dilution 1:1000; ab24648; Abcam, Shanghai, China) at 4 °C overnight, the PVDF membrane were washed in TBST buffer and incubated with secondary antibody for the anti-immune rabbit IgG (dilution 1:4000; LK2001; Sungene Biotech, Tianjin, China) conjugated with HRP. Antibody reacting bands were detected by means of ECL detection reagents. The β-tubulin was used as internal control, whose primary antibody (dilution 1:1000; KDM9003; Sungene Biotech, Tianjin, China) and secondary antibody for anti-immune mouse IgG-HRP (dilution 1:4000; LK2003; Sungene Biotech, Tianjin, China) were purchased from the Tianjin Sungene Biotech.