Protein Expression Analysis of Annexin Family

Preparation of protein extracts and western blot analysis were performed as previously described [17 (link)]. ANXA1 (37 kDa), ANXA2 (37 kDa), and ANXA4 (35 kDa) were respectively detected with rabbit polyclonal anti-ANXA1 (PA1006, Boster, Pleasanton, CA, USA), mouse monoclonal anti-ANXA2 (WH0000302M1, Sigma, Saint-Louis, MI, USA) and rabbit polyclonal anti-ANXA4 (PA1007-1, Boster, Pleasanton, CA, USA). Attempts to detect ANXA3 were performed using mouse monoclonal anti-ANXA3 (sc-390502, Santa Cruz Biotechnology, Dallas, TX, USA) or rabbit polyclonal anti-ANXA3 (PA1510, Boster, Pleasanton, CA, USA). GAPDH (loading control) was detected with a rabbit anti-GAPDH polyclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA). At least three independent experiments were performed for each ANX. Quantification of relative intensity of protein band was performed by ImageJ software. A Wilcoxon test was performed to identify putative statistical difference (p < 0.05) between values obtained for myoblasts and myotubes. These results are presented in Figure S1B.