Anti glyceraldehyde 3 phosphate dehydrogenase gapdh

After 48 h of cell transfection using Lipo8000™ Transfection Reagent (Beyotime), cells were collected in order to extract proteins from them using cell lysates (RIPA, Sangon Biotech, Shanghai, China; Cocktail, MCE, Shanghai, China). The concentration of protein in the total cell lysates was determined using the Enhanced BCA Protein Assay Kit (Beyotime). The protein was subjected to immunoblotting as described previously using the 12.5% Non-Closure SDS-PAGE Color Preparation kit (Sangon Biotech). The antibodies used were anti-Aurora B antibody (Huabio, Hangzhou, China 1:1000 for WB), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Zenbio, Chengdu, China, 1:5000 for WB), and HRP-conjugated anti-mouse (Huabio, 1:4000 for WB) or anti-rabbit immunoglobulin antibodies (Huabio, 1:50000 for WB). The expression of proteins was monitored using Immobilon Western Chemiluminescent HRP Substrate (Millipore, Burlington, MA, USA) reagent and visualized with an ECL detection system (Thermo Scientific, Waltham, MA, USA).

Western blot assay was performed to detect the protein levels of myogenic differentiation marker gene and the key proteins of IGF/PI3K/AKT signaling pathway, as previously described [22 (link)]. The primary antibodies were used are anti-myogenin (MyoG) (Biorbyt, Cambridge, UK; dilute 1:1000), anti-IGF2BP3 (ABclonal, Wuhan, China; 1:1000), anti-AKT1 (Cell Signaling Technology, Boston, MA, USA; 1:1000), anti-p-AKT1 (Cell Signaling Technology, Boston, MA, USA; 1:1000), anti-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ZenBio, Chengdu, China; 1:2000), and anti-β-tubulin (ZenBio, Chengdu, China; 1:2000). GAPDH and β-tubulin were used as endogenous controls. The relative protein levels were measured using Image J software.