Expression of Ca2+-handling proteins including STIM1, STIM2 and Orai1 during myogenic differentiation of C2C12 cells. A: Representative panel of Western blots for each protein of interest in lysates from C2C12 cells in an undifferentiated state and different stages of differentiation. B: Graph represents the average expression of three independent biological samples (mean ± S.E.M.) of C2C12 cells undergoing differentiation. All values were normalized to GAPDH, which was used as a loading control. C: A model for key proteins involved in the proper remodeling of myoblasts into myotubes, based on previous reports  and own data. The expression pattern of myogenin, taken from the study of MacLennan and co-workers , was up-regulated starting from day 1 of myoblast differentiation, which would mark the first wave of differentiation . Orai1, SERCA2a and RyR1 started to emerge at day 3, while cells exhibit a transient cardiac phenotype. Interestingly, we also observed an even shorter transient of STIM1 up-regulation, which seemed to decline at the same time as SERCA1 levels increased. Remarkably, while STIM1 was transiently up-regulated, STIM2 did not significantly alter and Orai1 continued to increase during differentiation. Materials and methods: Cells were collected 0, 1, 3 or 5 days after replacing myoblast culture medium with myotube differentiation medium [99, 113]. Cell lysates were analyzed for protein expression by Western blot and expression was each time normalized to GAPDH expression. Signals were detected with ECF and analyzed with ImageJ. Antibodies were from ProSci Inc., Poway, CA, USA (Orai1), Sigma, St Louis, MO, USA (STIM2 and GAPDH), Abnova, Taipei City, Taiwan (STIM1) and Thermo Scientific, Rockford, IL, USA (SERCA1 and RyR). The SERCA2a and IP3R1 antibodies are described elsewhere [114, 115]. Abbreviations: ECF, enhanced chemifluorescence; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IP3R, inositol 1,4,5-trisphosphate receptor; RyR, ryanodine receptor; SERCA, sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase; STIM, stromal interaction molecule.