Anti rabbit or anti mouse horseradish peroxidase conjugated secondary antibodies

Proteins of transfected myoblasts were extracted using lysis buffer and the concentration determined by a bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China). Total proteins (50 μg) were separated on a 12% SDS-PAGE, and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA). After blocked in 5% BSA blocking solution for 1 h at room temperature, the membrane was incubated overnight at 4°C with primary antibodies against MYOG (Biorbyt, Cambridge, UK; diluted 1:500) and MHC (DSHB, Iowa, USA; diluted 1:1,000). Subsequently, the membrane was developed with anti-rabbit or anti-mouse horseradish peroxidase conjugated secondary antibodies (Sigma-Aldrich, diluted 1:5,000). Protein bands were visualized using enhanced chemiluminescence (ECL) system (GE Healthcare, USA) and quantified with an ImageQuant LAS4000 system (Fujifilm, Tokyo, Japan).

Western blot analyses were performed using rabbit anti-RhsA (diluted 1:5,000, Genscript, custom polyclonal [14 (link)]), rabbit anti-FLAG (diluted 1:5,000, Sigma), rabbit anti-VSV-G (diluted 1:5,000; Sigma) and mouse anti-His₆ (diluted 1:5,000, Genscript) and detected with anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies (diluted 1:5,000; Sigma). Development of western blots was completed using chemiluminescent substrate (Clarity Max, Bio-Rad) and imaged with the ChemiDoc Imaging System (Bio-Rad).