Figure 2.

SOCE, co-immunoprecipitation of endogenous STIM1 with R429C, and expression levels of various proteins. (a) The Ca²⁺ in the SR of R429C-expressing myotubes was depleted by treatment with TG (2.5 μM) in the absence of extracellular Ca²⁺, and extracellular Ca²⁺ (2 mM) was then applied to the myotubes to induce SOCE. A representative trace for each group is shown. The boxed area was enlarged and dotted lines indicate the slopes in the rising phase of SOCE. The results are summarized as histograms for the area under the peaks (left-hand side) or for the slope (right-hand side). The experimental mean values were normalized to the control mean values. The values are presented as the mean ± s.e.m. for the number of myotubes shown in the parentheses in Table 1. (b) Co-immunoprecipitation assay of endogenous STIM1 with R429C was conducted using the lysate of R429C-expressing myotubes and anti-STIM1 antibody. Myotube lysate refers to the lysate of R429C-expressing myotubes. A representative result is presented. In the lane of myotube lysate, upper bands are CFP-R429Cs and lower bands are endogenous STIM1s. Degree of co-immunoprecipitated R429C to the corresponding total protein is presented as histograms in the right-hand side. *Significant difference was compared with ‘without anti-STIM1 Ab’ (p < 0.05). (c) The lysate of R429C-expressing myotubes was subjected to immunoblot assays with antibodies against the seven indicated proteins. α-actin was used as a loading control. A representative result is presented. The expression level of each protein normalized to the mean value of the control is presented as histograms on the right-hand side. The values in (b,c) are presented as the mean ± s.e.m. for the number of experiments shown in the parentheses of Supplementary Tables S3 and S4. *Significant difference compared with the control (p < 0.05). The blots that were cropped from different gels were grouped in (b,c), and the full-length blots are presented in Supplementary Figs. S4 and S5.