Fig. 1

DHPRα1 but not RyR1 is reduced in T-tubule striations of stac3 mutants. (A) Immunofluorescence labeling of WT sibling and stac3^−/− disassociated myotubes with anti-pan RyR (34c). (B) Mean immunofluorescence intensity of anti-RyR in stac3^−/− compared with WT siblings showing no difference in triadic RyR (t test, P = 0.89, n = 85 WT sibling, n = 50 stac3^−/−). a.u., arbitrary units. (C) Immunofluorescence labeling of WT sibling and stac3^−/− disassociated myotubes with mAb1 1A against a cytoplasmic region of DHPRα_1S (15). (D) Mean mAb1 1A labeling in WT siblings and stac3^−/− showing a decrease in triadic DHPRα_1S (t test, ***P < 0.0001, n = 216 WT sibling, n = 264 stac3^−/−). (E) Quantification of the mean of immunofluorescence labeling of anti-DHPRα_1S in stac3^−/− expressing stac3^NAM (NAM rescue) at triads compared with stac3^−/− expressing stac3^WT (WT rescue) (n = 75 stac3^−/−; stac3^WT, n = 53 stac3^−/−; stac3^NAM, t test, ****P < 0.0001). (F) Time course for FRAP of EGFP-DHPRα_1S expressed in WT siblings and stac3^−/− myofibers. Shown are EGFP-DHPRα_1S before (prebleach), after photobleaching (T = 0, 5, 34 min), and a maximum projection (stack) of T = 30 to 34 min (Right). (G) Mean quantification of the time course of FRAP in WT siblings (thick green line and circles) and stac3^−/− (thick red line and circles). Thin lines represent nonlinear regressions from individual traces of FRAPs from WT siblings (green) and stac3^−/− (red). The vertical thick green line depicts bleaching. (H) Histogram showing that the diffusion rate (D) of EGFP-DHPRα_1S is higher in stac3^−/− (t test, ***P < 0.0001, n = 33 stac3^−/−, n = 45 WT siblings). SEMs are indicated. (Scale bars, 2 μm.)