Figure S1.
Characterization of Cavin4 expression in zebrafish embryos and Cavin4−/− mouse muscle and C2C12 myotubes. (A) Western analysis of Cavin4b expression in 72-hpf WT and cavin4b−/− embryos, and adult zebrafish heart tissue. β-Dystroglycan (βDG) is shown as a protein loading control. Image is shown as a merge of chemiluminescent blot and colorimetric image. 15 µg lysate was loaded for each sample. Molecular weight marker (kilodaltons) is shown on left. (B) Whole-mount ISH of cavin4a and cavin4b in 24-hpf WT zebrafish embryos (images anterior to left in both dorsal and lateral view). Note the lack of notochord staining in dorsal view. Scale bars: 200 µm. (C) Whole-mount ISH of cavin4a and cavin4b in 24-hpf, 5-dpf, and 7-dpf WT zebrafish embryos highlighting a lack of cavin4a and cavin4b expression in the heart (arrows indicate positive control staining of anp, a cardiac-specific marker). Scale bars: 200 µm. (D) Confocal images showing subcellular localization of Clover-tagged Cavin4a, Cavin4b, and Cavin1a in muscle during zebrafish development (3, 5, 10, 14, and 19 dpf; top to bottom). Scale bars: 20 µm. Subsarcolemmal tubules were occasionally observed in older Cavin4b-Clover fish (inset, 14 dpf; bar: 10 µm). Tables show pairwise comparison of statistical differences observed for T-tubule to sarcolemmal fluorescence intensity ratios over the range of developmental stages (one-way ANOVA with multiple-comparison Tukey’s test). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. (E) Confocal images of CAVIN4 (with Phalloidin-Alexa488 counterstain, inset) in 3-d old WT and Cavin4−/− mouse skeletal muscle. Cavin4−/− #2 and #3 were used for analysis shown in Fig. 2. Scale bars: 10 µm. (F) Western analysis of CAVIN4 and BIN1 in 3-d old WT and Cavin4−/− mouse skeletal muscle (n = 3 each). Total sarcomeric actin (αACTIN) shown as a muscle-specific loading control. 25 µg lysate loaded for each sample. Molecular weight (in parentheses) is in kilodaltons. Molecular weight marker (kilodaltons) is shown on left. (G) qRT-PCR of Cavin4 and Bin1 expression (relative to 36B4) in WT (+/+) and Cavin4−/− (−/−) mouse skeletal muscle (n = 3 independent samples each for WT and Cavin4−/− performed in triplicate, two-tailed t test). (H) Confocal images (maximum projection) of CAVIN4 and CAV3 in WT and Cavin4−/− C2C12 myotubes. Inset shows boxed areas. Scale bars: 10 µm. (I) Western analysis of CAVIN4 and BIN1 in WT and Cavin4−/− C2C12 myotubes (n = 4 independent replicates). Total sarcomeric actin (αACTIN) shown as a muscle-specific loading control. 25 µg lysate was loaded for each sample. Molecular weight (in parentheses) is in kilodaltons. Molecular weight marker (kilodaltons) is shown on left. (J) qRT-PCR of Cavin4 and Bin1 expression (relative to 36B4) in WT (+/+) and Cavin4−/− (−/−) C2C12 myotubes (n = 3 independent samples each of WT and Cavin4−/− performed in triplicate, two-tailed t test). **, P ≤ 0.01; ***, P ≤ 0.001. Error bars represent mean ± SD.

Characterization of Cavin4 expression in zebrafish embryos and Cavin4−/− mouse muscle and C2C12 myotubes. (A) Western analysis of Cavin4b expression in 72-hpf WT and cavin4b−/− embryos, and adult zebrafish heart tissue. β-Dystroglycan (βDG) is shown as a protein loading control. Image is shown as a merge of chemiluminescent blot and colorimetric image. 15 µg lysate was loaded for each sample. Molecular weight marker (kilodaltons) is shown on left. (B) Whole-mount ISH of cavin4a and cavin4b in 24-hpf WT zebrafish embryos (images anterior to left in both dorsal and lateral view). Note the lack of notochord staining in dorsal view. Scale bars: 200 µm. (C) Whole-mount ISH of cavin4a and cavin4b in 24-hpf, 5-dpf, and 7-dpf WT zebrafish embryos highlighting a lack of cavin4a and cavin4b expression in the heart (arrows indicate positive control staining of anp, a cardiac-specific marker). Scale bars: 200 µm. (D) Confocal images showing subcellular localization of Clover-tagged Cavin4a, Cavin4b, and Cavin1a in muscle during zebrafish development (3, 5, 10, 14, and 19 dpf; top to bottom). Scale bars: 20 µm. Subsarcolemmal tubules were occasionally observed in older Cavin4b-Clover fish (inset, 14 dpf; bar: 10 µm). Tables show pairwise comparison of statistical differences observed for T-tubule to sarcolemmal fluorescence intensity ratios over the range of developmental stages (one-way ANOVA with multiple-comparison Tukey’s test). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. (E) Confocal images of CAVIN4 (with Phalloidin-Alexa488 counterstain, inset) in 3-d old WT and Cavin4−/− mouse skeletal muscle. Cavin4−/− #2 and #3 were used for analysis shown in Fig. 2. Scale bars: 10 µm. (F) Western analysis of CAVIN4 and BIN1 in 3-d old WT and Cavin4−/− mouse skeletal muscle (n = 3 each). Total sarcomeric actin (αACTIN) shown as a muscle-specific loading control. 25 µg lysate loaded for each sample. Molecular weight (in parentheses) is in kilodaltons. Molecular weight marker (kilodaltons) is shown on left. (G) qRT-PCR of Cavin4 and Bin1 expression (relative to 36B4) in WT (+/+) and Cavin4−/− (−/−) mouse skeletal muscle (n = 3 independent samples each for WT and Cavin4−/− performed in triplicate, two-tailed t test). (H) Confocal images (maximum projection) of CAVIN4 and CAV3 in WT and Cavin4−/− C2C12 myotubes. Inset shows boxed areas. Scale bars: 10 µm. (I) Western analysis of CAVIN4 and BIN1 in WT and Cavin4−/− C2C12 myotubes (n = 4 independent replicates). Total sarcomeric actin (αACTIN) shown as a muscle-specific loading control. 25 µg lysate was loaded for each sample. Molecular weight (in parentheses) is in kilodaltons. Molecular weight marker (kilodaltons) is shown on left. (J) qRT-PCR of Cavin4 and Bin1 expression (relative to 36B4) in WT (+/+) and Cavin4−/− (−/−) C2C12 myotubes (n = 3 independent samples each of WT and Cavin4−/− performed in triplicate, two-tailed t test). **, P ≤ 0.01; ***, P ≤ 0.001. Error bars represent mean ± SD.

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