caveolin Search Results


human caveolin 1 elisa kit  (Shanghai Korain Biotech Co Ltd)
90
Shanghai Korain Biotech Co Ltd human caveolin 1 elisa kit
Human Caveolin 1 Elisa Kit, supplied by Shanghai Korain Biotech Co Ltd, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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caveolin 1  (Bioss)
94
Bioss caveolin 1
Caveolin 1, supplied by Bioss, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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cav 1 d46g3 rabbit antibody  (Cell Signaling Technology Inc)
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Cell Signaling Technology Inc cav 1 d46g3 rabbit antibody
Cav 1 D46g3 Rabbit Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rabbit polyclonal anti caveolin 1  (Cell Signaling Technology Inc)
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Cell Signaling Technology Inc rabbit polyclonal anti caveolin 1
Reagents used in this study
Rabbit Polyclonal Anti Caveolin 1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti cav1  (Cell Signaling Technology Inc)
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Cell Signaling Technology Inc anti cav1
Reagents used in this study
Anti Cav1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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caveolin 1  (Santa Cruz Biotechnology)
96
Santa Cruz Biotechnology caveolin 1
Reagents used in this study
Caveolin 1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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antibody mouse anti cav3 monoclonal santa cruz sc 5310  (Santa Cruz Biotechnology)
94
Santa Cruz Biotechnology antibody mouse anti cav3 monoclonal santa cruz sc 5310
Figure 1. Generation of syndapin III KO mice. (a) Murine syndapin III domain structure and putative remaining peptide upon syndapin III exon 5 and 6 deletion. (b) Generation of syndapin III KO mice. Scheme of the syndapin III gene comprising 11 exons (coding exon parts in black) and of targeting vector and strategy of Southern blot analyses. Homologous recombination (homologous regions in dark grey and black) resulted in floxed exons 5 and 6. (c–e) Merges of MIPs of NIH3T3 cells transfected with GFP (c), GFP-syndapin III1-70 peptide (putatively remaining upon KO; composed of aa1-70 of syndapin III and five unrelated aa resulting from the frameshift caused by exon 5,6 deletion) (d) and GFP-syndapin III F-BAR (e), respectively. Cotransfected plasma membrane-targeted mCherry (mCherryF) served as internal control for a membrane-bound protein. In (e), the GFP channel of a GFP-syndapin III F-BAR-transfected cell is shown in addition to the merge to visualize the tubular structures induced by syndapin III F-BAR. The inset in (e) shows an enlargement of the boxed area. Bars, 10 mm. For enlarged images see Figure 1—figure supplement 1. (f–h) Immunoblotting analyses of fractionations of transfected HEK293 cells showing that whereas plasma membrane-targeted mCherry (f–h, lower panel) and GFP-syndapin III F-BAR (h, upper panel) are readily detectable in the crude membrane fraction P2, both GFP and GFP-syndapin III1-70 are not (f,g, upper panels). (i) MIPs of NIH3T3 cells transfected with GFP-syndapin III F-BAR coexpressing mCherry-syndapin III1-70 showing undisturbed membrane localization, self-assembly and membrane tubulation abilities of GFP-syndapin III F-BAR. Bars, 10 mm. For enlarged images see Figure 1—figure supplement 2. (j) Southern blot analysis of exemplary ES cell clones (G10, transgenic; H7, WT). (k) Genotyping of the offspring of heterozygous mating identifies all possible genotypes. (l) Normal frequency of genotypes and genders of syndapin III KO mice. (m,n) RT-PCRs on heart (m) and skeletal muscle cDNA (n). (o) Immunoblottings of tissue homogenates (50 mg each) show the lack of syndapin III in KO tissues. GAPDH and b-actin, controls. (p–r) Immunofluorescence analyses of syndapin III and <t>cav3</t> in transversal skeletal muscle sections from WT (p) and syndapin III KO mice (r) and quantitative colocalization analyses (q) in ROIs placed at plasma membrane (n = 240 ROIs from eight images) and intracellular areas (n = 160 ROIs from eight images) in confocal stacks of images of transversal sections of WT skeletal muscles. Data, mean ± SEM. Statistical significance, Mann-Whitney U test. Bars, 100 mm. Figure 1 continued on next page
Antibody Mouse Anti Cav3 Monoclonal Santa Cruz Sc 5310, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rabbit anti p cav 1  (Proteintech)
95
Proteintech rabbit anti p cav 1
Figure 1. Generation of syndapin III KO mice. (a) Murine syndapin III domain structure and putative remaining peptide upon syndapin III exon 5 and 6 deletion. (b) Generation of syndapin III KO mice. Scheme of the syndapin III gene comprising 11 exons (coding exon parts in black) and of targeting vector and strategy of Southern blot analyses. Homologous recombination (homologous regions in dark grey and black) resulted in floxed exons 5 and 6. (c–e) Merges of MIPs of NIH3T3 cells transfected with GFP (c), GFP-syndapin III1-70 peptide (putatively remaining upon KO; composed of aa1-70 of syndapin III and five unrelated aa resulting from the frameshift caused by exon 5,6 deletion) (d) and GFP-syndapin III F-BAR (e), respectively. Cotransfected plasma membrane-targeted mCherry (mCherryF) served as internal control for a membrane-bound protein. In (e), the GFP channel of a GFP-syndapin III F-BAR-transfected cell is shown in addition to the merge to visualize the tubular structures induced by syndapin III F-BAR. The inset in (e) shows an enlargement of the boxed area. Bars, 10 mm. For enlarged images see Figure 1—figure supplement 1. (f–h) Immunoblotting analyses of fractionations of transfected HEK293 cells showing that whereas plasma membrane-targeted mCherry (f–h, lower panel) and GFP-syndapin III F-BAR (h, upper panel) are readily detectable in the crude membrane fraction P2, both GFP and GFP-syndapin III1-70 are not (f,g, upper panels). (i) MIPs of NIH3T3 cells transfected with GFP-syndapin III F-BAR coexpressing mCherry-syndapin III1-70 showing undisturbed membrane localization, self-assembly and membrane tubulation abilities of GFP-syndapin III F-BAR. Bars, 10 mm. For enlarged images see Figure 1—figure supplement 2. (j) Southern blot analysis of exemplary ES cell clones (G10, transgenic; H7, WT). (k) Genotyping of the offspring of heterozygous mating identifies all possible genotypes. (l) Normal frequency of genotypes and genders of syndapin III KO mice. (m,n) RT-PCRs on heart (m) and skeletal muscle cDNA (n). (o) Immunoblottings of tissue homogenates (50 mg each) show the lack of syndapin III in KO tissues. GAPDH and b-actin, controls. (p–r) Immunofluorescence analyses of syndapin III and <t>cav3</t> in transversal skeletal muscle sections from WT (p) and syndapin III KO mice (r) and quantitative colocalization analyses (q) in ROIs placed at plasma membrane (n = 240 ROIs from eight images) and intracellular areas (n = 160 ROIs from eight images) in confocal stacks of images of transversal sections of WT skeletal muscles. Data, mean ± SEM. Statistical significance, Mann-Whitney U test. Bars, 100 mm. Figure 1 continued on next page
Rabbit Anti P Cav 1, supplied by Proteintech, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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cav1 2  (Santa Cruz Biotechnology)
97
Santa Cruz Biotechnology cav1 2
Figure 1. Generation of syndapin III KO mice. (a) Murine syndapin III domain structure and putative remaining peptide upon syndapin III exon 5 and 6 deletion. (b) Generation of syndapin III KO mice. Scheme of the syndapin III gene comprising 11 exons (coding exon parts in black) and of targeting vector and strategy of Southern blot analyses. Homologous recombination (homologous regions in dark grey and black) resulted in floxed exons 5 and 6. (c–e) Merges of MIPs of NIH3T3 cells transfected with GFP (c), GFP-syndapin III1-70 peptide (putatively remaining upon KO; composed of aa1-70 of syndapin III and five unrelated aa resulting from the frameshift caused by exon 5,6 deletion) (d) and GFP-syndapin III F-BAR (e), respectively. Cotransfected plasma membrane-targeted mCherry (mCherryF) served as internal control for a membrane-bound protein. In (e), the GFP channel of a GFP-syndapin III F-BAR-transfected cell is shown in addition to the merge to visualize the tubular structures induced by syndapin III F-BAR. The inset in (e) shows an enlargement of the boxed area. Bars, 10 mm. For enlarged images see Figure 1—figure supplement 1. (f–h) Immunoblotting analyses of fractionations of transfected HEK293 cells showing that whereas plasma membrane-targeted mCherry (f–h, lower panel) and GFP-syndapin III F-BAR (h, upper panel) are readily detectable in the crude membrane fraction P2, both GFP and GFP-syndapin III1-70 are not (f,g, upper panels). (i) MIPs of NIH3T3 cells transfected with GFP-syndapin III F-BAR coexpressing mCherry-syndapin III1-70 showing undisturbed membrane localization, self-assembly and membrane tubulation abilities of GFP-syndapin III F-BAR. Bars, 10 mm. For enlarged images see Figure 1—figure supplement 2. (j) Southern blot analysis of exemplary ES cell clones (G10, transgenic; H7, WT). (k) Genotyping of the offspring of heterozygous mating identifies all possible genotypes. (l) Normal frequency of genotypes and genders of syndapin III KO mice. (m,n) RT-PCRs on heart (m) and skeletal muscle cDNA (n). (o) Immunoblottings of tissue homogenates (50 mg each) show the lack of syndapin III in KO tissues. GAPDH and b-actin, controls. (p–r) Immunofluorescence analyses of syndapin III and <t>cav3</t> in transversal skeletal muscle sections from WT (p) and syndapin III KO mice (r) and quantitative colocalization analyses (q) in ROIs placed at plasma membrane (n = 240 ROIs from eight images) and intracellular areas (n = 160 ROIs from eight images) in confocal stacks of images of transversal sections of WT skeletal muscles. Data, mean ± SEM. Statistical significance, Mann-Whitney U test. Bars, 100 mm. Figure 1 continued on next page
Cav1 2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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cst anti c src  (Cell Signaling Technology Inc)
94
Cell Signaling Technology Inc cst anti c src
Figure 1. Generation of syndapin III KO mice. (a) Murine syndapin III domain structure and putative remaining peptide upon syndapin III exon 5 and 6 deletion. (b) Generation of syndapin III KO mice. Scheme of the syndapin III gene comprising 11 exons (coding exon parts in black) and of targeting vector and strategy of Southern blot analyses. Homologous recombination (homologous regions in dark grey and black) resulted in floxed exons 5 and 6. (c–e) Merges of MIPs of NIH3T3 cells transfected with GFP (c), GFP-syndapin III1-70 peptide (putatively remaining upon KO; composed of aa1-70 of syndapin III and five unrelated aa resulting from the frameshift caused by exon 5,6 deletion) (d) and GFP-syndapin III F-BAR (e), respectively. Cotransfected plasma membrane-targeted mCherry (mCherryF) served as internal control for a membrane-bound protein. In (e), the GFP channel of a GFP-syndapin III F-BAR-transfected cell is shown in addition to the merge to visualize the tubular structures induced by syndapin III F-BAR. The inset in (e) shows an enlargement of the boxed area. Bars, 10 mm. For enlarged images see Figure 1—figure supplement 1. (f–h) Immunoblotting analyses of fractionations of transfected HEK293 cells showing that whereas plasma membrane-targeted mCherry (f–h, lower panel) and GFP-syndapin III F-BAR (h, upper panel) are readily detectable in the crude membrane fraction P2, both GFP and GFP-syndapin III1-70 are not (f,g, upper panels). (i) MIPs of NIH3T3 cells transfected with GFP-syndapin III F-BAR coexpressing mCherry-syndapin III1-70 showing undisturbed membrane localization, self-assembly and membrane tubulation abilities of GFP-syndapin III F-BAR. Bars, 10 mm. For enlarged images see Figure 1—figure supplement 2. (j) Southern blot analysis of exemplary ES cell clones (G10, transgenic; H7, WT). (k) Genotyping of the offspring of heterozygous mating identifies all possible genotypes. (l) Normal frequency of genotypes and genders of syndapin III KO mice. (m,n) RT-PCRs on heart (m) and skeletal muscle cDNA (n). (o) Immunoblottings of tissue homogenates (50 mg each) show the lack of syndapin III in KO tissues. GAPDH and b-actin, controls. (p–r) Immunofluorescence analyses of syndapin III and <t>cav3</t> in transversal skeletal muscle sections from WT (p) and syndapin III KO mice (r) and quantitative colocalization analyses (q) in ROIs placed at plasma membrane (n = 240 ROIs from eight images) and intracellular areas (n = 160 ROIs from eight images) in confocal stacks of images of transversal sections of WT skeletal muscles. Data, mean ± SEM. Statistical significance, Mann-Whitney U test. Bars, 100 mm. Figure 1 continued on next page
Cst Anti C Src, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mcherry caveolin c  (Addgene inc)
93
Addgene inc mcherry caveolin c
Figure 1. Generation of syndapin III KO mice. (a) Murine syndapin III domain structure and putative remaining peptide upon syndapin III exon 5 and 6 deletion. (b) Generation of syndapin III KO mice. Scheme of the syndapin III gene comprising 11 exons (coding exon parts in black) and of targeting vector and strategy of Southern blot analyses. Homologous recombination (homologous regions in dark grey and black) resulted in floxed exons 5 and 6. (c–e) Merges of MIPs of NIH3T3 cells transfected with GFP (c), GFP-syndapin III1-70 peptide (putatively remaining upon KO; composed of aa1-70 of syndapin III and five unrelated aa resulting from the frameshift caused by exon 5,6 deletion) (d) and GFP-syndapin III F-BAR (e), respectively. Cotransfected plasma membrane-targeted mCherry (mCherryF) served as internal control for a membrane-bound protein. In (e), the GFP channel of a GFP-syndapin III F-BAR-transfected cell is shown in addition to the merge to visualize the tubular structures induced by syndapin III F-BAR. The inset in (e) shows an enlargement of the boxed area. Bars, 10 mm. For enlarged images see Figure 1—figure supplement 1. (f–h) Immunoblotting analyses of fractionations of transfected HEK293 cells showing that whereas plasma membrane-targeted mCherry (f–h, lower panel) and GFP-syndapin III F-BAR (h, upper panel) are readily detectable in the crude membrane fraction P2, both GFP and GFP-syndapin III1-70 are not (f,g, upper panels). (i) MIPs of NIH3T3 cells transfected with GFP-syndapin III F-BAR coexpressing mCherry-syndapin III1-70 showing undisturbed membrane localization, self-assembly and membrane tubulation abilities of GFP-syndapin III F-BAR. Bars, 10 mm. For enlarged images see Figure 1—figure supplement 2. (j) Southern blot analysis of exemplary ES cell clones (G10, transgenic; H7, WT). (k) Genotyping of the offspring of heterozygous mating identifies all possible genotypes. (l) Normal frequency of genotypes and genders of syndapin III KO mice. (m,n) RT-PCRs on heart (m) and skeletal muscle cDNA (n). (o) Immunoblottings of tissue homogenates (50 mg each) show the lack of syndapin III in KO tissues. GAPDH and b-actin, controls. (p–r) Immunofluorescence analyses of syndapin III and <t>cav3</t> in transversal skeletal muscle sections from WT (p) and syndapin III KO mice (r) and quantitative colocalization analyses (q) in ROIs placed at plasma membrane (n = 240 ROIs from eight images) and intracellular areas (n = 160 ROIs from eight images) in confocal stacks of images of transversal sections of WT skeletal muscles. Data, mean ± SEM. Statistical significance, Mann-Whitney U test. Bars, 100 mm. Figure 1 continued on next page
Mcherry Caveolin C, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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cav 1 sirna  (Santa Cruz Biotechnology)
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Santa Cruz Biotechnology cav 1 sirna
Figure 1. Generation of syndapin III KO mice. (a) Murine syndapin III domain structure and putative remaining peptide upon syndapin III exon 5 and 6 deletion. (b) Generation of syndapin III KO mice. Scheme of the syndapin III gene comprising 11 exons (coding exon parts in black) and of targeting vector and strategy of Southern blot analyses. Homologous recombination (homologous regions in dark grey and black) resulted in floxed exons 5 and 6. (c–e) Merges of MIPs of NIH3T3 cells transfected with GFP (c), GFP-syndapin III1-70 peptide (putatively remaining upon KO; composed of aa1-70 of syndapin III and five unrelated aa resulting from the frameshift caused by exon 5,6 deletion) (d) and GFP-syndapin III F-BAR (e), respectively. Cotransfected plasma membrane-targeted mCherry (mCherryF) served as internal control for a membrane-bound protein. In (e), the GFP channel of a GFP-syndapin III F-BAR-transfected cell is shown in addition to the merge to visualize the tubular structures induced by syndapin III F-BAR. The inset in (e) shows an enlargement of the boxed area. Bars, 10 mm. For enlarged images see Figure 1—figure supplement 1. (f–h) Immunoblotting analyses of fractionations of transfected HEK293 cells showing that whereas plasma membrane-targeted mCherry (f–h, lower panel) and GFP-syndapin III F-BAR (h, upper panel) are readily detectable in the crude membrane fraction P2, both GFP and GFP-syndapin III1-70 are not (f,g, upper panels). (i) MIPs of NIH3T3 cells transfected with GFP-syndapin III F-BAR coexpressing mCherry-syndapin III1-70 showing undisturbed membrane localization, self-assembly and membrane tubulation abilities of GFP-syndapin III F-BAR. Bars, 10 mm. For enlarged images see Figure 1—figure supplement 2. (j) Southern blot analysis of exemplary ES cell clones (G10, transgenic; H7, WT). (k) Genotyping of the offspring of heterozygous mating identifies all possible genotypes. (l) Normal frequency of genotypes and genders of syndapin III KO mice. (m,n) RT-PCRs on heart (m) and skeletal muscle cDNA (n). (o) Immunoblottings of tissue homogenates (50 mg each) show the lack of syndapin III in KO tissues. GAPDH and b-actin, controls. (p–r) Immunofluorescence analyses of syndapin III and <t>cav3</t> in transversal skeletal muscle sections from WT (p) and syndapin III KO mice (r) and quantitative colocalization analyses (q) in ROIs placed at plasma membrane (n = 240 ROIs from eight images) and intracellular areas (n = 160 ROIs from eight images) in confocal stacks of images of transversal sections of WT skeletal muscles. Data, mean ± SEM. Statistical significance, Mann-Whitney U test. Bars, 100 mm. Figure 1 continued on next page
Cav 1 Sirna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


Reagents used in this study

Journal: Genes & Development

Article Title: Fatty acid uptake activates an AXL–CAV1–β-catenin axis to drive melanoma progression

doi: 10.1101/gad.351985.124

Figure Lengend Snippet: Reagents used in this study

Article Snippet: Rabbit polyclonal anti-Caveolin-1 , Cell Signaling Technology , 3238 , AB_2072166.

Techniques: Software, Transduction, Virus, Subcloning, Bacteria, Recombinant, Bradford Assay, Protease Inhibitor, RNA Extraction, Reporter Assay, Bicinchoninic Acid Protein Assay, Quantitative RT-PCR, Negative Control

Figure 1. Generation of syndapin III KO mice. (a) Murine syndapin III domain structure and putative remaining peptide upon syndapin III exon 5 and 6 deletion. (b) Generation of syndapin III KO mice. Scheme of the syndapin III gene comprising 11 exons (coding exon parts in black) and of targeting vector and strategy of Southern blot analyses. Homologous recombination (homologous regions in dark grey and black) resulted in floxed exons 5 and 6. (c–e) Merges of MIPs of NIH3T3 cells transfected with GFP (c), GFP-syndapin III1-70 peptide (putatively remaining upon KO; composed of aa1-70 of syndapin III and five unrelated aa resulting from the frameshift caused by exon 5,6 deletion) (d) and GFP-syndapin III F-BAR (e), respectively. Cotransfected plasma membrane-targeted mCherry (mCherryF) served as internal control for a membrane-bound protein. In (e), the GFP channel of a GFP-syndapin III F-BAR-transfected cell is shown in addition to the merge to visualize the tubular structures induced by syndapin III F-BAR. The inset in (e) shows an enlargement of the boxed area. Bars, 10 mm. For enlarged images see Figure 1—figure supplement 1. (f–h) Immunoblotting analyses of fractionations of transfected HEK293 cells showing that whereas plasma membrane-targeted mCherry (f–h, lower panel) and GFP-syndapin III F-BAR (h, upper panel) are readily detectable in the crude membrane fraction P2, both GFP and GFP-syndapin III1-70 are not (f,g, upper panels). (i) MIPs of NIH3T3 cells transfected with GFP-syndapin III F-BAR coexpressing mCherry-syndapin III1-70 showing undisturbed membrane localization, self-assembly and membrane tubulation abilities of GFP-syndapin III F-BAR. Bars, 10 mm. For enlarged images see Figure 1—figure supplement 2. (j) Southern blot analysis of exemplary ES cell clones (G10, transgenic; H7, WT). (k) Genotyping of the offspring of heterozygous mating identifies all possible genotypes. (l) Normal frequency of genotypes and genders of syndapin III KO mice. (m,n) RT-PCRs on heart (m) and skeletal muscle cDNA (n). (o) Immunoblottings of tissue homogenates (50 mg each) show the lack of syndapin III in KO tissues. GAPDH and b-actin, controls. (p–r) Immunofluorescence analyses of syndapin III and cav3 in transversal skeletal muscle sections from WT (p) and syndapin III KO mice (r) and quantitative colocalization analyses (q) in ROIs placed at plasma membrane (n = 240 ROIs from eight images) and intracellular areas (n = 160 ROIs from eight images) in confocal stacks of images of transversal sections of WT skeletal muscles. Data, mean ± SEM. Statistical significance, Mann-Whitney U test. Bars, 100 mm. Figure 1 continued on next page

Journal: eLife

Article Title: Deciphering caveolar functions by syndapin III KO-mediated impairment of caveolar invagination

doi: 10.7554/elife.29854

Figure Lengend Snippet: Figure 1. Generation of syndapin III KO mice. (a) Murine syndapin III domain structure and putative remaining peptide upon syndapin III exon 5 and 6 deletion. (b) Generation of syndapin III KO mice. Scheme of the syndapin III gene comprising 11 exons (coding exon parts in black) and of targeting vector and strategy of Southern blot analyses. Homologous recombination (homologous regions in dark grey and black) resulted in floxed exons 5 and 6. (c–e) Merges of MIPs of NIH3T3 cells transfected with GFP (c), GFP-syndapin III1-70 peptide (putatively remaining upon KO; composed of aa1-70 of syndapin III and five unrelated aa resulting from the frameshift caused by exon 5,6 deletion) (d) and GFP-syndapin III F-BAR (e), respectively. Cotransfected plasma membrane-targeted mCherry (mCherryF) served as internal control for a membrane-bound protein. In (e), the GFP channel of a GFP-syndapin III F-BAR-transfected cell is shown in addition to the merge to visualize the tubular structures induced by syndapin III F-BAR. The inset in (e) shows an enlargement of the boxed area. Bars, 10 mm. For enlarged images see Figure 1—figure supplement 1. (f–h) Immunoblotting analyses of fractionations of transfected HEK293 cells showing that whereas plasma membrane-targeted mCherry (f–h, lower panel) and GFP-syndapin III F-BAR (h, upper panel) are readily detectable in the crude membrane fraction P2, both GFP and GFP-syndapin III1-70 are not (f,g, upper panels). (i) MIPs of NIH3T3 cells transfected with GFP-syndapin III F-BAR coexpressing mCherry-syndapin III1-70 showing undisturbed membrane localization, self-assembly and membrane tubulation abilities of GFP-syndapin III F-BAR. Bars, 10 mm. For enlarged images see Figure 1—figure supplement 2. (j) Southern blot analysis of exemplary ES cell clones (G10, transgenic; H7, WT). (k) Genotyping of the offspring of heterozygous mating identifies all possible genotypes. (l) Normal frequency of genotypes and genders of syndapin III KO mice. (m,n) RT-PCRs on heart (m) and skeletal muscle cDNA (n). (o) Immunoblottings of tissue homogenates (50 mg each) show the lack of syndapin III in KO tissues. GAPDH and b-actin, controls. (p–r) Immunofluorescence analyses of syndapin III and cav3 in transversal skeletal muscle sections from WT (p) and syndapin III KO mice (r) and quantitative colocalization analyses (q) in ROIs placed at plasma membrane (n = 240 ROIs from eight images) and intracellular areas (n = 160 ROIs from eight images) in confocal stacks of images of transversal sections of WT skeletal muscles. Data, mean ± SEM. Statistical significance, Mann-Whitney U test. Bars, 100 mm. Figure 1 continued on next page

Article Snippet: Koch et al. (2012), Histochem Cell Biol 138: 215-230., Qualmann and Kelly (2000), J Cell Biol 148:1047–1062 anti-syndapin I, II, III 1:1000 (western blot); 1:50 (FRIL) Antibody goat anti-GAPDH (polyclonal) Santa Cruz (Dallas, Texas) sc48167 AB_1563046 1:1000 Antibody mouse anti-cav3 (monoclonal ) Santa Cruz sc-5310, AB_626814 1:500 (western blot), 1:50 (IHC), 1:50 (FRIL) Antibody goat anti-cav3 (polyclonal) Santa Cruz sc-7665 AB_637945 1:500 (western blot), 1:50 (IHC) Antibody mouse anti-cav1 (monoclonal) Santa Cruz sc-53564, AB_628859 1:500 (western blot), 1:50 (IHC), 1:50 (FRIL) Antibody rabbit anti-cav1 (polyclonal) Santa Cruz sc-894 AB_2072042 1:1000 (western blot), 1:200 (IHC), 1:50 (FRIL) Antibody rabbit anti-IRTK (polyclonal) Santa Cruz sc-710, AB_631106 1:1000 (western blot) Antibody mouse anti-CAVIN-1 (monoclonal) BD Bioscience (Franklin Lakes, New Jersey) 611258, AB_398788 1:50 (FRIL) Antibody mouse anti-PDI (monoclonal) Stressgene (Farmingdale, New York) ADI-SPA-891, AB_10615355 1:1000 (western blot) Antibody mouse anti-b-actin (monoclonal) Sigma A5441, AB_476744 1:5000 (western blot) Antibody mouse anti-b-tubulin (monoclonal) Sigma T4026, AB_477577 1:1500 (western blot) Antibody rabbit anti-CAVIN-1 (polyclonal) Proteintech (Rosemont, Illinois) 18892–1-AP, AB_10596795 1:1000 (western blot), 1:100 (IHC), 1:50 (FRIL) Antibody rabbit anti-CAVIN-3 (polyclonal) Proteintech 16250–1-AP AB_2171894 1:1000 (western blot) Antibody goat anti-CAVIN-2 (polyclonal) R & D Systems (Minneapolis, Minnesota) AF5759 AB_2269901 1:200 (western blot) Antibody rabbit anti-CAVIN-4 (polyclonal) Sigma HPA020987 AB_1853080 1:400 (western blot) Continued on next page Seemann et al. eLife 2017;6:e29854.

Techniques: Plasmid Preparation, Southern Blot, Homologous Recombination, Transfection, Clinical Proteomics, Membrane, Control, Western Blot, Clone Assay, Transgenic Assay, Immunofluorescence, Muscles, MANN-WHITNEY

Figure 3. Syndapin III KO leads to impairments in the formation of cav3-coated caveolae. (a,b) Details of wide-field TEM images of anti-syndapin III immunogold-labeled P-faces of freeze-fractured plasma membranes of cardiomyocytes (CDMC) from WT (a) and syndapin III KO mice (b). (c) Blinded, quantitative evaluations of the anti-syndapin III labeling distribution on full areas of freeze-fractured membranes. (a–c) WT, 15.8 mm2 from 20 images; KO, 27 mm2 from 20 images (two independent cardiomyocyte preparations each pooled from two animals/genotype). (d-i’) Electron micrographs of coimmunolabeled control surfaces (d,e) and P-faces of WT (h,h’,h’’; for the picture, from which the details in h’ and h’’ were taken, see Figure 3— figure supplement 1) and syndapin III KO cells (i,i’) as well as blinded quantitative evaluations of labeling densities (f,g; n = 20 images each condition) demonstrating the specificity of the labelings at the P-face of WT cardiomyocytes. Syndapin III (15 nm gold, red labels) is present at caveolae highlighted by anti-cav3 labeling (10 nm gold, blue labels). Labelings at caveolae are marked by black arrows, at shallow indentations by grey arrows and at flat membrane areas by grey arrowheads. Bars, 100 nm. For non-color-marked EM micrographs see Figure 3—figure supplement 2. (j) Analyses of the fractions of caveolae-like profiles (deep and shallow) and of non-caveolar invaginations that were either unlabeled or labeled for cav3, syndapin III and both, respectively (n = 92 WT invaginations). (k,l) Blinded quantitative analyses of the relative densities of caveolae (deep, 70 nm in diameter invaginations formally confirmed as caveolae by anti-cav3 labeling) (k), and of shallow indentations (l), which also were anti-cav3-positive. n = 20 images each; in total, 132 cav3-positive structures were scored. (m) Densities of the (rare) non-caveolar invaginations in WT and syndapin III KO cardiomyocytes Figure 3 continued on next page

Journal: eLife

Article Title: Deciphering caveolar functions by syndapin III KO-mediated impairment of caveolar invagination

doi: 10.7554/elife.29854

Figure Lengend Snippet: Figure 3. Syndapin III KO leads to impairments in the formation of cav3-coated caveolae. (a,b) Details of wide-field TEM images of anti-syndapin III immunogold-labeled P-faces of freeze-fractured plasma membranes of cardiomyocytes (CDMC) from WT (a) and syndapin III KO mice (b). (c) Blinded, quantitative evaluations of the anti-syndapin III labeling distribution on full areas of freeze-fractured membranes. (a–c) WT, 15.8 mm2 from 20 images; KO, 27 mm2 from 20 images (two independent cardiomyocyte preparations each pooled from two animals/genotype). (d-i’) Electron micrographs of coimmunolabeled control surfaces (d,e) and P-faces of WT (h,h’,h’’; for the picture, from which the details in h’ and h’’ were taken, see Figure 3— figure supplement 1) and syndapin III KO cells (i,i’) as well as blinded quantitative evaluations of labeling densities (f,g; n = 20 images each condition) demonstrating the specificity of the labelings at the P-face of WT cardiomyocytes. Syndapin III (15 nm gold, red labels) is present at caveolae highlighted by anti-cav3 labeling (10 nm gold, blue labels). Labelings at caveolae are marked by black arrows, at shallow indentations by grey arrows and at flat membrane areas by grey arrowheads. Bars, 100 nm. For non-color-marked EM micrographs see Figure 3—figure supplement 2. (j) Analyses of the fractions of caveolae-like profiles (deep and shallow) and of non-caveolar invaginations that were either unlabeled or labeled for cav3, syndapin III and both, respectively (n = 92 WT invaginations). (k,l) Blinded quantitative analyses of the relative densities of caveolae (deep, 70 nm in diameter invaginations formally confirmed as caveolae by anti-cav3 labeling) (k), and of shallow indentations (l), which also were anti-cav3-positive. n = 20 images each; in total, 132 cav3-positive structures were scored. (m) Densities of the (rare) non-caveolar invaginations in WT and syndapin III KO cardiomyocytes Figure 3 continued on next page

Article Snippet: Koch et al. (2012), Histochem Cell Biol 138: 215-230., Qualmann and Kelly (2000), J Cell Biol 148:1047–1062 anti-syndapin I, II, III 1:1000 (western blot); 1:50 (FRIL) Antibody goat anti-GAPDH (polyclonal) Santa Cruz (Dallas, Texas) sc48167 AB_1563046 1:1000 Antibody mouse anti-cav3 (monoclonal ) Santa Cruz sc-5310, AB_626814 1:500 (western blot), 1:50 (IHC), 1:50 (FRIL) Antibody goat anti-cav3 (polyclonal) Santa Cruz sc-7665 AB_637945 1:500 (western blot), 1:50 (IHC) Antibody mouse anti-cav1 (monoclonal) Santa Cruz sc-53564, AB_628859 1:500 (western blot), 1:50 (IHC), 1:50 (FRIL) Antibody rabbit anti-cav1 (polyclonal) Santa Cruz sc-894 AB_2072042 1:1000 (western blot), 1:200 (IHC), 1:50 (FRIL) Antibody rabbit anti-IRTK (polyclonal) Santa Cruz sc-710, AB_631106 1:1000 (western blot) Antibody mouse anti-CAVIN-1 (monoclonal) BD Bioscience (Franklin Lakes, New Jersey) 611258, AB_398788 1:50 (FRIL) Antibody mouse anti-PDI (monoclonal) Stressgene (Farmingdale, New York) ADI-SPA-891, AB_10615355 1:1000 (western blot) Antibody mouse anti-b-actin (monoclonal) Sigma A5441, AB_476744 1:5000 (western blot) Antibody mouse anti-b-tubulin (monoclonal) Sigma T4026, AB_477577 1:1500 (western blot) Antibody rabbit anti-CAVIN-1 (polyclonal) Proteintech (Rosemont, Illinois) 18892–1-AP, AB_10596795 1:1000 (western blot), 1:100 (IHC), 1:50 (FRIL) Antibody rabbit anti-CAVIN-3 (polyclonal) Proteintech 16250–1-AP AB_2171894 1:1000 (western blot) Antibody goat anti-CAVIN-2 (polyclonal) R & D Systems (Minneapolis, Minnesota) AF5759 AB_2269901 1:200 (western blot) Antibody rabbit anti-CAVIN-4 (polyclonal) Sigma HPA020987 AB_1853080 1:400 (western blot) Continued on next page Seemann et al. eLife 2017;6:e29854.

Techniques: Labeling, Clinical Proteomics, Control, Membrane

Figure 4. Impairment of caveolar invagination by syndapin III KO does not lead to dissociation of CAVIN-1 from the plasma membrane. (a,b) Quantitative western blot analyses of homogenates of hearts and skeletal muscles from WT and syndapin III KO mice showing that the levels of the important cav3 coat component CAVIN-1 are unaffected. (c-f) Quantitative western blot analyses of homogenates of hearts (c,d) and skeletal muscles (e,f) from WT and syndapin III KO mice addressing components suggested to play roles in caveolae formation that may be redundant or related to the critical role of syndapin III in caveolar invagination (normalized to GAPDH). Data, mean ± SEM. N = 12 each genotype. (g,h) Electron micrographs of anti-CAVIN-1 immunogold labeling (green labels) of freeze-fractured cardiomyocytes isolated from WT (g) and syndapin III KO mice (h) (in combination with a low concentration immunolabeling of cav3 (blue labels) to prove that indeed a membrane of a (cav3-positive) cardiomyocyte is examined). Note that CAVIN-1’s membrane association and its ability to form clusters is not impaired in syndapin III KO cells. Examples of clustered labeling of CAVIN-1 at (cav3-positive) deeply invaginated caveolae are marked by black arrows, at shallow indentations by grey arrows and at flat membrane areas by grey arrowheads. For non-color-marked EM micrographs see Figure 4—figure supplement 1. Bars, 100 nm. (i,j) Quantitative analyses of the labeling Figure 4 continued on next page

Journal: eLife

Article Title: Deciphering caveolar functions by syndapin III KO-mediated impairment of caveolar invagination

doi: 10.7554/elife.29854

Figure Lengend Snippet: Figure 4. Impairment of caveolar invagination by syndapin III KO does not lead to dissociation of CAVIN-1 from the plasma membrane. (a,b) Quantitative western blot analyses of homogenates of hearts and skeletal muscles from WT and syndapin III KO mice showing that the levels of the important cav3 coat component CAVIN-1 are unaffected. (c-f) Quantitative western blot analyses of homogenates of hearts (c,d) and skeletal muscles (e,f) from WT and syndapin III KO mice addressing components suggested to play roles in caveolae formation that may be redundant or related to the critical role of syndapin III in caveolar invagination (normalized to GAPDH). Data, mean ± SEM. N = 12 each genotype. (g,h) Electron micrographs of anti-CAVIN-1 immunogold labeling (green labels) of freeze-fractured cardiomyocytes isolated from WT (g) and syndapin III KO mice (h) (in combination with a low concentration immunolabeling of cav3 (blue labels) to prove that indeed a membrane of a (cav3-positive) cardiomyocyte is examined). Note that CAVIN-1’s membrane association and its ability to form clusters is not impaired in syndapin III KO cells. Examples of clustered labeling of CAVIN-1 at (cav3-positive) deeply invaginated caveolae are marked by black arrows, at shallow indentations by grey arrows and at flat membrane areas by grey arrowheads. For non-color-marked EM micrographs see Figure 4—figure supplement 1. Bars, 100 nm. (i,j) Quantitative analyses of the labeling Figure 4 continued on next page

Article Snippet: Koch et al. (2012), Histochem Cell Biol 138: 215-230., Qualmann and Kelly (2000), J Cell Biol 148:1047–1062 anti-syndapin I, II, III 1:1000 (western blot); 1:50 (FRIL) Antibody goat anti-GAPDH (polyclonal) Santa Cruz (Dallas, Texas) sc48167 AB_1563046 1:1000 Antibody mouse anti-cav3 (monoclonal ) Santa Cruz sc-5310, AB_626814 1:500 (western blot), 1:50 (IHC), 1:50 (FRIL) Antibody goat anti-cav3 (polyclonal) Santa Cruz sc-7665 AB_637945 1:500 (western blot), 1:50 (IHC) Antibody mouse anti-cav1 (monoclonal) Santa Cruz sc-53564, AB_628859 1:500 (western blot), 1:50 (IHC), 1:50 (FRIL) Antibody rabbit anti-cav1 (polyclonal) Santa Cruz sc-894 AB_2072042 1:1000 (western blot), 1:200 (IHC), 1:50 (FRIL) Antibody rabbit anti-IRTK (polyclonal) Santa Cruz sc-710, AB_631106 1:1000 (western blot) Antibody mouse anti-CAVIN-1 (monoclonal) BD Bioscience (Franklin Lakes, New Jersey) 611258, AB_398788 1:50 (FRIL) Antibody mouse anti-PDI (monoclonal) Stressgene (Farmingdale, New York) ADI-SPA-891, AB_10615355 1:1000 (western blot) Antibody mouse anti-b-actin (monoclonal) Sigma A5441, AB_476744 1:5000 (western blot) Antibody mouse anti-b-tubulin (monoclonal) Sigma T4026, AB_477577 1:1500 (western blot) Antibody rabbit anti-CAVIN-1 (polyclonal) Proteintech (Rosemont, Illinois) 18892–1-AP, AB_10596795 1:1000 (western blot), 1:100 (IHC), 1:50 (FRIL) Antibody rabbit anti-CAVIN-3 (polyclonal) Proteintech 16250–1-AP AB_2171894 1:1000 (western blot) Antibody goat anti-CAVIN-2 (polyclonal) R & D Systems (Minneapolis, Minnesota) AF5759 AB_2269901 1:200 (western blot) Antibody rabbit anti-CAVIN-4 (polyclonal) Sigma HPA020987 AB_1853080 1:400 (western blot) Continued on next page Seemann et al. eLife 2017;6:e29854.

Techniques: Clinical Proteomics, Membrane, Western Blot, Muscles, Labeling, Isolation, Concentration Assay, Immunolabeling

Figure 5. Syndapin III shapes liposomes into tubules with caveolar diameters and localizes to the rim of cav3 coats. (a–c) Analyses of tubules induced by incubating liposomes with syndapin III (a,b) and GST (c), respectively, by freeze-fracturing/TEM (a) as well as by cryo-TEM (b,c). (d,e) Quantitative analyses of tubule diameter distributions (d) and averages of diameters (e) induced by syndapin III and GST-syndapin III, respectively. Data in (e), mean +SEM. (d,e) n= 50 (GST-syndapin III) and n = 38 (syndapin III) freeze-fractured tubuli. (f) High resolution, 80 kV top view of a deeply invaginated caveolar structure of a WT cardiomyocyte immunolabeled for syndapin III (15 nm gold) and cav3 (10 nm gold). Bar, 50 nm. (g,h) Corresponding 120 kV tomogram images (g) and views from a 3D-reconstruction (h) show that syndapin III is at the edge of the cav3 coat (34 caveolae analyzed; two full 3D- segmentations; invaginated membrane, green; immunogold labels cav3, yellow; syndapin III, red). Bars in f–h), 50 nm. DOI: https://doi.org/10.7554/eLife.29854.016 The following source data is available for figure 5:

Journal: eLife

Article Title: Deciphering caveolar functions by syndapin III KO-mediated impairment of caveolar invagination

doi: 10.7554/elife.29854

Figure Lengend Snippet: Figure 5. Syndapin III shapes liposomes into tubules with caveolar diameters and localizes to the rim of cav3 coats. (a–c) Analyses of tubules induced by incubating liposomes with syndapin III (a,b) and GST (c), respectively, by freeze-fracturing/TEM (a) as well as by cryo-TEM (b,c). (d,e) Quantitative analyses of tubule diameter distributions (d) and averages of diameters (e) induced by syndapin III and GST-syndapin III, respectively. Data in (e), mean +SEM. (d,e) n= 50 (GST-syndapin III) and n = 38 (syndapin III) freeze-fractured tubuli. (f) High resolution, 80 kV top view of a deeply invaginated caveolar structure of a WT cardiomyocyte immunolabeled for syndapin III (15 nm gold) and cav3 (10 nm gold). Bar, 50 nm. (g,h) Corresponding 120 kV tomogram images (g) and views from a 3D-reconstruction (h) show that syndapin III is at the edge of the cav3 coat (34 caveolae analyzed; two full 3D- segmentations; invaginated membrane, green; immunogold labels cav3, yellow; syndapin III, red). Bars in f–h), 50 nm. DOI: https://doi.org/10.7554/eLife.29854.016 The following source data is available for figure 5:

Article Snippet: Koch et al. (2012), Histochem Cell Biol 138: 215-230., Qualmann and Kelly (2000), J Cell Biol 148:1047–1062 anti-syndapin I, II, III 1:1000 (western blot); 1:50 (FRIL) Antibody goat anti-GAPDH (polyclonal) Santa Cruz (Dallas, Texas) sc48167 AB_1563046 1:1000 Antibody mouse anti-cav3 (monoclonal ) Santa Cruz sc-5310, AB_626814 1:500 (western blot), 1:50 (IHC), 1:50 (FRIL) Antibody goat anti-cav3 (polyclonal) Santa Cruz sc-7665 AB_637945 1:500 (western blot), 1:50 (IHC) Antibody mouse anti-cav1 (monoclonal) Santa Cruz sc-53564, AB_628859 1:500 (western blot), 1:50 (IHC), 1:50 (FRIL) Antibody rabbit anti-cav1 (polyclonal) Santa Cruz sc-894 AB_2072042 1:1000 (western blot), 1:200 (IHC), 1:50 (FRIL) Antibody rabbit anti-IRTK (polyclonal) Santa Cruz sc-710, AB_631106 1:1000 (western blot) Antibody mouse anti-CAVIN-1 (monoclonal) BD Bioscience (Franklin Lakes, New Jersey) 611258, AB_398788 1:50 (FRIL) Antibody mouse anti-PDI (monoclonal) Stressgene (Farmingdale, New York) ADI-SPA-891, AB_10615355 1:1000 (western blot) Antibody mouse anti-b-actin (monoclonal) Sigma A5441, AB_476744 1:5000 (western blot) Antibody mouse anti-b-tubulin (monoclonal) Sigma T4026, AB_477577 1:1500 (western blot) Antibody rabbit anti-CAVIN-1 (polyclonal) Proteintech (Rosemont, Illinois) 18892–1-AP, AB_10596795 1:1000 (western blot), 1:100 (IHC), 1:50 (FRIL) Antibody rabbit anti-CAVIN-3 (polyclonal) Proteintech 16250–1-AP AB_2171894 1:1000 (western blot) Antibody goat anti-CAVIN-2 (polyclonal) R & D Systems (Minneapolis, Minnesota) AF5759 AB_2269901 1:200 (western blot) Antibody rabbit anti-CAVIN-4 (polyclonal) Sigma HPA020987 AB_1853080 1:400 (western blot) Continued on next page Seemann et al. eLife 2017;6:e29854.

Techniques: Liposomes, Immunolabeling, Membrane

Figure 6. Syndapin III is involved in the organization of cav3-containing membrane domains. (a) Coprecipitation of endogenous cav3 from heart lysates with the indicated immobilized GST-fusion proteins of syndapin III. (b,c) GFP-syndapin III F-BAR domain but not GFP clusters with cav3 at the membrane of primary cardiomyocytes. Boxed areas, higher magnification insets. Bars, 10 mm. (d–k) Syndapin III KO changes the biophysical properties of cav3-containing DRMs. Immunoblottings of cav3 and syndapin III (d–g) and proteins representing Golgi (GM130), cytosol (GAPDH), plasma membrane (IRTK) and ER (PDI) (j,k) in TritonX-100-resistant membrane preparations from heart and skeletal muscles from WT (d,e) and syndapin III KO mice (f,g,j,k). Quantitative analyses (h,i) demonstrate the shift of cav3-containing TritonX-100-resistant membranes from fractions F4 +F5 to F6 +F7 in heart (d,f,h) and from F3 +F4 to F5 +F6 in skeletal muscle (e,g,i) upon syndapin III KO. Hearts, n = 3 each genotype; skeletal muscles, n = 9 each genotype. Data, mean ± SEM. Statistical significances, two-way ANOVA and Bonferroni post-test. For individual comparisons of WT and KO fractions see Figure 6—figure supplement 1. DOI: https://doi.org/10.7554/eLife.29854.020 The following source data and figure supplement are available for figure 6:

Journal: eLife

Article Title: Deciphering caveolar functions by syndapin III KO-mediated impairment of caveolar invagination

doi: 10.7554/elife.29854

Figure Lengend Snippet: Figure 6. Syndapin III is involved in the organization of cav3-containing membrane domains. (a) Coprecipitation of endogenous cav3 from heart lysates with the indicated immobilized GST-fusion proteins of syndapin III. (b,c) GFP-syndapin III F-BAR domain but not GFP clusters with cav3 at the membrane of primary cardiomyocytes. Boxed areas, higher magnification insets. Bars, 10 mm. (d–k) Syndapin III KO changes the biophysical properties of cav3-containing DRMs. Immunoblottings of cav3 and syndapin III (d–g) and proteins representing Golgi (GM130), cytosol (GAPDH), plasma membrane (IRTK) and ER (PDI) (j,k) in TritonX-100-resistant membrane preparations from heart and skeletal muscles from WT (d,e) and syndapin III KO mice (f,g,j,k). Quantitative analyses (h,i) demonstrate the shift of cav3-containing TritonX-100-resistant membranes from fractions F4 +F5 to F6 +F7 in heart (d,f,h) and from F3 +F4 to F5 +F6 in skeletal muscle (e,g,i) upon syndapin III KO. Hearts, n = 3 each genotype; skeletal muscles, n = 9 each genotype. Data, mean ± SEM. Statistical significances, two-way ANOVA and Bonferroni post-test. For individual comparisons of WT and KO fractions see Figure 6—figure supplement 1. DOI: https://doi.org/10.7554/eLife.29854.020 The following source data and figure supplement are available for figure 6:

Article Snippet: Koch et al. (2012), Histochem Cell Biol 138: 215-230., Qualmann and Kelly (2000), J Cell Biol 148:1047–1062 anti-syndapin I, II, III 1:1000 (western blot); 1:50 (FRIL) Antibody goat anti-GAPDH (polyclonal) Santa Cruz (Dallas, Texas) sc48167 AB_1563046 1:1000 Antibody mouse anti-cav3 (monoclonal ) Santa Cruz sc-5310, AB_626814 1:500 (western blot), 1:50 (IHC), 1:50 (FRIL) Antibody goat anti-cav3 (polyclonal) Santa Cruz sc-7665 AB_637945 1:500 (western blot), 1:50 (IHC) Antibody mouse anti-cav1 (monoclonal) Santa Cruz sc-53564, AB_628859 1:500 (western blot), 1:50 (IHC), 1:50 (FRIL) Antibody rabbit anti-cav1 (polyclonal) Santa Cruz sc-894 AB_2072042 1:1000 (western blot), 1:200 (IHC), 1:50 (FRIL) Antibody rabbit anti-IRTK (polyclonal) Santa Cruz sc-710, AB_631106 1:1000 (western blot) Antibody mouse anti-CAVIN-1 (monoclonal) BD Bioscience (Franklin Lakes, New Jersey) 611258, AB_398788 1:50 (FRIL) Antibody mouse anti-PDI (monoclonal) Stressgene (Farmingdale, New York) ADI-SPA-891, AB_10615355 1:1000 (western blot) Antibody mouse anti-b-actin (monoclonal) Sigma A5441, AB_476744 1:5000 (western blot) Antibody mouse anti-b-tubulin (monoclonal) Sigma T4026, AB_477577 1:1500 (western blot) Antibody rabbit anti-CAVIN-1 (polyclonal) Proteintech (Rosemont, Illinois) 18892–1-AP, AB_10596795 1:1000 (western blot), 1:100 (IHC), 1:50 (FRIL) Antibody rabbit anti-CAVIN-3 (polyclonal) Proteintech 16250–1-AP AB_2171894 1:1000 (western blot) Antibody goat anti-CAVIN-2 (polyclonal) R & D Systems (Minneapolis, Minnesota) AF5759 AB_2269901 1:200 (western blot) Antibody rabbit anti-CAVIN-4 (polyclonal) Sigma HPA020987 AB_1853080 1:400 (western blot) Continued on next page Seemann et al. eLife 2017;6:e29854.

Techniques: Membrane, Clinical Proteomics, Muscles

Figure 7. Not all described cav3 loss-of-function phenotypes are found upon syndapin III KO and may thus not reflect impairments of caveolar invagination. (a,b) Quantitative immunoblot analyses of ERK1/2 and phosphoERK1/2 in heart homogenates of WT and syndapin III KO mice (normalized to WT) show no alteration of MAPK signaling (pERK1/2/ERK1/2 signals). Data represent mean ± SEM; n = 12 each. (c–f) Unchanged subcellular localization of both dystrophin and b-dystroglycan in sections of skeletal muscles from syndapin III KO mice. Bars, 50 mm. (g–j) Levels of dystrophin and b-dystroglycan are unchanged in heart and skeletal muscle homogenates upon syndapin III KO (normalized to WT). Data represent mean ± SEM; n = 12 each. DOI: https://doi.org/10.7554/eLife.29854.023 The following source data is available for figure 7:

Journal: eLife

Article Title: Deciphering caveolar functions by syndapin III KO-mediated impairment of caveolar invagination

doi: 10.7554/elife.29854

Figure Lengend Snippet: Figure 7. Not all described cav3 loss-of-function phenotypes are found upon syndapin III KO and may thus not reflect impairments of caveolar invagination. (a,b) Quantitative immunoblot analyses of ERK1/2 and phosphoERK1/2 in heart homogenates of WT and syndapin III KO mice (normalized to WT) show no alteration of MAPK signaling (pERK1/2/ERK1/2 signals). Data represent mean ± SEM; n = 12 each. (c–f) Unchanged subcellular localization of both dystrophin and b-dystroglycan in sections of skeletal muscles from syndapin III KO mice. Bars, 50 mm. (g–j) Levels of dystrophin and b-dystroglycan are unchanged in heart and skeletal muscle homogenates upon syndapin III KO (normalized to WT). Data represent mean ± SEM; n = 12 each. DOI: https://doi.org/10.7554/eLife.29854.023 The following source data is available for figure 7:

Article Snippet: Koch et al. (2012), Histochem Cell Biol 138: 215-230., Qualmann and Kelly (2000), J Cell Biol 148:1047–1062 anti-syndapin I, II, III 1:1000 (western blot); 1:50 (FRIL) Antibody goat anti-GAPDH (polyclonal) Santa Cruz (Dallas, Texas) sc48167 AB_1563046 1:1000 Antibody mouse anti-cav3 (monoclonal ) Santa Cruz sc-5310, AB_626814 1:500 (western blot), 1:50 (IHC), 1:50 (FRIL) Antibody goat anti-cav3 (polyclonal) Santa Cruz sc-7665 AB_637945 1:500 (western blot), 1:50 (IHC) Antibody mouse anti-cav1 (monoclonal) Santa Cruz sc-53564, AB_628859 1:500 (western blot), 1:50 (IHC), 1:50 (FRIL) Antibody rabbit anti-cav1 (polyclonal) Santa Cruz sc-894 AB_2072042 1:1000 (western blot), 1:200 (IHC), 1:50 (FRIL) Antibody rabbit anti-IRTK (polyclonal) Santa Cruz sc-710, AB_631106 1:1000 (western blot) Antibody mouse anti-CAVIN-1 (monoclonal) BD Bioscience (Franklin Lakes, New Jersey) 611258, AB_398788 1:50 (FRIL) Antibody mouse anti-PDI (monoclonal) Stressgene (Farmingdale, New York) ADI-SPA-891, AB_10615355 1:1000 (western blot) Antibody mouse anti-b-actin (monoclonal) Sigma A5441, AB_476744 1:5000 (western blot) Antibody mouse anti-b-tubulin (monoclonal) Sigma T4026, AB_477577 1:1500 (western blot) Antibody rabbit anti-CAVIN-1 (polyclonal) Proteintech (Rosemont, Illinois) 18892–1-AP, AB_10596795 1:1000 (western blot), 1:100 (IHC), 1:50 (FRIL) Antibody rabbit anti-CAVIN-3 (polyclonal) Proteintech 16250–1-AP AB_2171894 1:1000 (western blot) Antibody goat anti-CAVIN-2 (polyclonal) R & D Systems (Minneapolis, Minnesota) AF5759 AB_2269901 1:200 (western blot) Antibody rabbit anti-CAVIN-4 (polyclonal) Sigma HPA020987 AB_1853080 1:400 (western blot) Continued on next page Seemann et al. eLife 2017;6:e29854.

Techniques: Western Blot, Muscles

Figure 8. Syndapin III-mediated caveolar invagination counterpoises membrane tensions and thereby ensures cell integrity. (a,b) Anti-cav3-labeled P-faces of freeze-fractured plasma membranes of cardiomyocytes from WT mice incubated for 5 min in isoosmotic (iso) (a) and hypoosmotic conditions (hypo; hypo15 buffer) (b). Bars, 200 nm. (c,d) A dramatic reduction of deeply invaginated caveolar structures was observed upon the induced, cell swelling-mediated rise in membrane tension caused by hypo conditions. (e–h) Quantitation of the anti-cav3 labeling distribution in total and in relations to deeply invaginated, shallow and flat membrane topologies (relative to isoosmotic condition). Data (c–h), mean ± SEM. iso, 148.8 mm2 membrane area from 43 images, 251 caveolar invaginations; hypo, 124.6 mm2 membrane area from 36 images, 66 caveolar invaginations, two independent cardiomyocyte preparations and assays. Statistical significance, two-tailed Student’s t test. For related examinations of caveolar flattening upon induction of membrane tensions in NIH3T3 cells please see Figure 8—figure supplement 1. (i–o) Trypan Blue assays with WT (i–k) and syndapin III KO cardiomyocytes (l–n) subjected to membrane tensions mimicked by mild hypoosmotic stress (5 min hypo30; j,m) and stronger hypoosmotic stress (5 min hypo15; k,n) unveiling a higher vulnerability of syndapin III KO cells under conditions increasing membrane tensions. Note the increased abundancy of Trypan Blue-positive, ruptured cardiomyocytes upon hypoosmotic stress. Bar in n (for i–n), 50 mm. (o) Quantitative data, mean ± SEM of 18 blinded experiments (about 100 cells each assay) from six independent preparations of cardiomyocytes/genotype. One-way Anova with Tukey’s post-test. DOI: https://doi.org/10.7554/eLife.29854.025 The following source data and figure supplement are available for figure 8:

Journal: eLife

Article Title: Deciphering caveolar functions by syndapin III KO-mediated impairment of caveolar invagination

doi: 10.7554/elife.29854

Figure Lengend Snippet: Figure 8. Syndapin III-mediated caveolar invagination counterpoises membrane tensions and thereby ensures cell integrity. (a,b) Anti-cav3-labeled P-faces of freeze-fractured plasma membranes of cardiomyocytes from WT mice incubated for 5 min in isoosmotic (iso) (a) and hypoosmotic conditions (hypo; hypo15 buffer) (b). Bars, 200 nm. (c,d) A dramatic reduction of deeply invaginated caveolar structures was observed upon the induced, cell swelling-mediated rise in membrane tension caused by hypo conditions. (e–h) Quantitation of the anti-cav3 labeling distribution in total and in relations to deeply invaginated, shallow and flat membrane topologies (relative to isoosmotic condition). Data (c–h), mean ± SEM. iso, 148.8 mm2 membrane area from 43 images, 251 caveolar invaginations; hypo, 124.6 mm2 membrane area from 36 images, 66 caveolar invaginations, two independent cardiomyocyte preparations and assays. Statistical significance, two-tailed Student’s t test. For related examinations of caveolar flattening upon induction of membrane tensions in NIH3T3 cells please see Figure 8—figure supplement 1. (i–o) Trypan Blue assays with WT (i–k) and syndapin III KO cardiomyocytes (l–n) subjected to membrane tensions mimicked by mild hypoosmotic stress (5 min hypo30; j,m) and stronger hypoosmotic stress (5 min hypo15; k,n) unveiling a higher vulnerability of syndapin III KO cells under conditions increasing membrane tensions. Note the increased abundancy of Trypan Blue-positive, ruptured cardiomyocytes upon hypoosmotic stress. Bar in n (for i–n), 50 mm. (o) Quantitative data, mean ± SEM of 18 blinded experiments (about 100 cells each assay) from six independent preparations of cardiomyocytes/genotype. One-way Anova with Tukey’s post-test. DOI: https://doi.org/10.7554/eLife.29854.025 The following source data and figure supplement are available for figure 8:

Article Snippet: Koch et al. (2012), Histochem Cell Biol 138: 215-230., Qualmann and Kelly (2000), J Cell Biol 148:1047–1062 anti-syndapin I, II, III 1:1000 (western blot); 1:50 (FRIL) Antibody goat anti-GAPDH (polyclonal) Santa Cruz (Dallas, Texas) sc48167 AB_1563046 1:1000 Antibody mouse anti-cav3 (monoclonal ) Santa Cruz sc-5310, AB_626814 1:500 (western blot), 1:50 (IHC), 1:50 (FRIL) Antibody goat anti-cav3 (polyclonal) Santa Cruz sc-7665 AB_637945 1:500 (western blot), 1:50 (IHC) Antibody mouse anti-cav1 (monoclonal) Santa Cruz sc-53564, AB_628859 1:500 (western blot), 1:50 (IHC), 1:50 (FRIL) Antibody rabbit anti-cav1 (polyclonal) Santa Cruz sc-894 AB_2072042 1:1000 (western blot), 1:200 (IHC), 1:50 (FRIL) Antibody rabbit anti-IRTK (polyclonal) Santa Cruz sc-710, AB_631106 1:1000 (western blot) Antibody mouse anti-CAVIN-1 (monoclonal) BD Bioscience (Franklin Lakes, New Jersey) 611258, AB_398788 1:50 (FRIL) Antibody mouse anti-PDI (monoclonal) Stressgene (Farmingdale, New York) ADI-SPA-891, AB_10615355 1:1000 (western blot) Antibody mouse anti-b-actin (monoclonal) Sigma A5441, AB_476744 1:5000 (western blot) Antibody mouse anti-b-tubulin (monoclonal) Sigma T4026, AB_477577 1:1500 (western blot) Antibody rabbit anti-CAVIN-1 (polyclonal) Proteintech (Rosemont, Illinois) 18892–1-AP, AB_10596795 1:1000 (western blot), 1:100 (IHC), 1:50 (FRIL) Antibody rabbit anti-CAVIN-3 (polyclonal) Proteintech 16250–1-AP AB_2171894 1:1000 (western blot) Antibody goat anti-CAVIN-2 (polyclonal) R & D Systems (Minneapolis, Minnesota) AF5759 AB_2269901 1:200 (western blot) Antibody rabbit anti-CAVIN-4 (polyclonal) Sigma HPA020987 AB_1853080 1:400 (western blot) Continued on next page Seemann et al. eLife 2017;6:e29854.

Techniques: Membrane, Labeling, Clinical Proteomics, Incubation, Quantitation Assay, Two Tailed Test

Figure 10. Syndapin III KO causes impairments in caveolar invagination in skeletal muscle. (a,b) Double-immunogold labeled, freeze-fractured skeletal muscles of WT and syndapin III KO mice (syndapin III, 15 nm gold; cav3, 10 nm gold) show that caveolar invagination is impaired in tissues of syndapin III KO mice. Bar, 200 nm. (c) Analyses of the fractions of caveolar-like profiles (deep and shallow) and non-caveolar invaginations labeled for cav3, syndapin III and both in freeze-fractured muscles. In total, 291 invaginations from WT skeletal muscles were evaluated. (d) Densities of the (extremely rare) non-caveolar invaginations in WT and syndapin III KO cardiomyocytes (low abundance of such structures, in total only n = 2 (WT) and n = 3 (KO) found; n.s.). (e–j) Colocalization analyses based on confocal images of longitudinal sections of skeletal muscles immunostained for cav3 and syndapin III. (e,f) Example images. Encircled is a ROI for colocalization analyses at puncta (e); (f) shows magnification and (g) shows the corresponding Pearson correlation coefficient (n = 60 ROIs). (h–j) Quantitative analyses of anti-cav3 immunofluorescence signals at the plasma membrane ((h); n = 230 ROIs each genotype in transversal sections), in intracellular volumes ((i); n = 160 ROIs each genotype in transversal sections) and in cav3 puncta ((j); n = 60 each genotype in longitudinal sections). (k) Quantitative immunoblotting of TritonX-100-insoluble material showed no differences in cav3 and cav1 levels upon syndapin III KO. 25 mg protein loaded each. WT, 12 animals; KO, 12 animals. (l) Quantitative analyses of immunogold-labeled, freeze- fractured skeletal muscle fibers showing no difference in anti-cav3 labeling density between WT and syndapin III KO muscles at the plasma membrane. (m,n) Example of 3D surface rendering (using IMARIS) of confocal image stacks of the anti-cav3 immunolabeling shown in overview (e) for determination Figure 10 continued on next page

Journal: eLife

Article Title: Deciphering caveolar functions by syndapin III KO-mediated impairment of caveolar invagination

doi: 10.7554/elife.29854

Figure Lengend Snippet: Figure 10. Syndapin III KO causes impairments in caveolar invagination in skeletal muscle. (a,b) Double-immunogold labeled, freeze-fractured skeletal muscles of WT and syndapin III KO mice (syndapin III, 15 nm gold; cav3, 10 nm gold) show that caveolar invagination is impaired in tissues of syndapin III KO mice. Bar, 200 nm. (c) Analyses of the fractions of caveolar-like profiles (deep and shallow) and non-caveolar invaginations labeled for cav3, syndapin III and both in freeze-fractured muscles. In total, 291 invaginations from WT skeletal muscles were evaluated. (d) Densities of the (extremely rare) non-caveolar invaginations in WT and syndapin III KO cardiomyocytes (low abundance of such structures, in total only n = 2 (WT) and n = 3 (KO) found; n.s.). (e–j) Colocalization analyses based on confocal images of longitudinal sections of skeletal muscles immunostained for cav3 and syndapin III. (e,f) Example images. Encircled is a ROI for colocalization analyses at puncta (e); (f) shows magnification and (g) shows the corresponding Pearson correlation coefficient (n = 60 ROIs). (h–j) Quantitative analyses of anti-cav3 immunofluorescence signals at the plasma membrane ((h); n = 230 ROIs each genotype in transversal sections), in intracellular volumes ((i); n = 160 ROIs each genotype in transversal sections) and in cav3 puncta ((j); n = 60 each genotype in longitudinal sections). (k) Quantitative immunoblotting of TritonX-100-insoluble material showed no differences in cav3 and cav1 levels upon syndapin III KO. 25 mg protein loaded each. WT, 12 animals; KO, 12 animals. (l) Quantitative analyses of immunogold-labeled, freeze- fractured skeletal muscle fibers showing no difference in anti-cav3 labeling density between WT and syndapin III KO muscles at the plasma membrane. (m,n) Example of 3D surface rendering (using IMARIS) of confocal image stacks of the anti-cav3 immunolabeling shown in overview (e) for determination Figure 10 continued on next page

Article Snippet: Koch et al. (2012), Histochem Cell Biol 138: 215-230., Qualmann and Kelly (2000), J Cell Biol 148:1047–1062 anti-syndapin I, II, III 1:1000 (western blot); 1:50 (FRIL) Antibody goat anti-GAPDH (polyclonal) Santa Cruz (Dallas, Texas) sc48167 AB_1563046 1:1000 Antibody mouse anti-cav3 (monoclonal ) Santa Cruz sc-5310, AB_626814 1:500 (western blot), 1:50 (IHC), 1:50 (FRIL) Antibody goat anti-cav3 (polyclonal) Santa Cruz sc-7665 AB_637945 1:500 (western blot), 1:50 (IHC) Antibody mouse anti-cav1 (monoclonal) Santa Cruz sc-53564, AB_628859 1:500 (western blot), 1:50 (IHC), 1:50 (FRIL) Antibody rabbit anti-cav1 (polyclonal) Santa Cruz sc-894 AB_2072042 1:1000 (western blot), 1:200 (IHC), 1:50 (FRIL) Antibody rabbit anti-IRTK (polyclonal) Santa Cruz sc-710, AB_631106 1:1000 (western blot) Antibody mouse anti-CAVIN-1 (monoclonal) BD Bioscience (Franklin Lakes, New Jersey) 611258, AB_398788 1:50 (FRIL) Antibody mouse anti-PDI (monoclonal) Stressgene (Farmingdale, New York) ADI-SPA-891, AB_10615355 1:1000 (western blot) Antibody mouse anti-b-actin (monoclonal) Sigma A5441, AB_476744 1:5000 (western blot) Antibody mouse anti-b-tubulin (monoclonal) Sigma T4026, AB_477577 1:1500 (western blot) Antibody rabbit anti-CAVIN-1 (polyclonal) Proteintech (Rosemont, Illinois) 18892–1-AP, AB_10596795 1:1000 (western blot), 1:100 (IHC), 1:50 (FRIL) Antibody rabbit anti-CAVIN-3 (polyclonal) Proteintech 16250–1-AP AB_2171894 1:1000 (western blot) Antibody goat anti-CAVIN-2 (polyclonal) R & D Systems (Minneapolis, Minnesota) AF5759 AB_2269901 1:200 (western blot) Antibody rabbit anti-CAVIN-4 (polyclonal) Sigma HPA020987 AB_1853080 1:400 (western blot) Continued on next page Seemann et al. eLife 2017;6:e29854.

Techniques: Labeling, Muscles, Immunofluorescence, Clinical Proteomics, Membrane, Western Blot, Immunolabeling

Figure 11. Syndapin III KO leads to skeletal muscle phenotypes reminiscent of clinical symptoms found in patients with myopathies associated with CAV3 mutation. (a–f) Histological examinations of cryosections of musculus gastrocnemius from >64 weeks old WT and syndapin III KO mice (three animals each). Whereas no clear signs of cellular damage or disintegration were observed by H&E staining under untrained conditions (a,b), syndapin III KO mice displayed a higher frequency of detached nuclei upon training (c), arrows). (e,f) Acidic phosphatase stainings clearly demonstrate necrotic events (asterisks in c) and e). Bars, 50 mm. (g,h) Quantitative evaluations of the frequency of detached nuclei. (i,j) Percental distribution of fiber cross sectional areas (in 50 mm2 intervals) in WT and syndapin III KO muscles. Arrows highlight areas of the caliber spectra with striking differences (black, more frequent in syndapin III KO; grey, more frequent in WT). (k,l) Quantitation of the mean cross sectional areas of muscle fibers in trained and untrained mice of both genotypes. N-numbers (g–l), untrained WT, 849; untrained KO, 952; trained WT, 949; trained KO, 1244 fibers. Data, mean (i,j); error bars omitted for clarity) and mean ± SEM (g,h,k,l), respectively. Statistical significance, 2-tailed Student’s t-test. DOI: https://doi.org/10.7554/eLife.29854.032

Journal: eLife

Article Title: Deciphering caveolar functions by syndapin III KO-mediated impairment of caveolar invagination

doi: 10.7554/elife.29854

Figure Lengend Snippet: Figure 11. Syndapin III KO leads to skeletal muscle phenotypes reminiscent of clinical symptoms found in patients with myopathies associated with CAV3 mutation. (a–f) Histological examinations of cryosections of musculus gastrocnemius from >64 weeks old WT and syndapin III KO mice (three animals each). Whereas no clear signs of cellular damage or disintegration were observed by H&E staining under untrained conditions (a,b), syndapin III KO mice displayed a higher frequency of detached nuclei upon training (c), arrows). (e,f) Acidic phosphatase stainings clearly demonstrate necrotic events (asterisks in c) and e). Bars, 50 mm. (g,h) Quantitative evaluations of the frequency of detached nuclei. (i,j) Percental distribution of fiber cross sectional areas (in 50 mm2 intervals) in WT and syndapin III KO muscles. Arrows highlight areas of the caliber spectra with striking differences (black, more frequent in syndapin III KO; grey, more frequent in WT). (k,l) Quantitation of the mean cross sectional areas of muscle fibers in trained and untrained mice of both genotypes. N-numbers (g–l), untrained WT, 849; untrained KO, 952; trained WT, 949; trained KO, 1244 fibers. Data, mean (i,j); error bars omitted for clarity) and mean ± SEM (g,h,k,l), respectively. Statistical significance, 2-tailed Student’s t-test. DOI: https://doi.org/10.7554/eLife.29854.032

Article Snippet: Koch et al. (2012), Histochem Cell Biol 138: 215-230., Qualmann and Kelly (2000), J Cell Biol 148:1047–1062 anti-syndapin I, II, III 1:1000 (western blot); 1:50 (FRIL) Antibody goat anti-GAPDH (polyclonal) Santa Cruz (Dallas, Texas) sc48167 AB_1563046 1:1000 Antibody mouse anti-cav3 (monoclonal ) Santa Cruz sc-5310, AB_626814 1:500 (western blot), 1:50 (IHC), 1:50 (FRIL) Antibody goat anti-cav3 (polyclonal) Santa Cruz sc-7665 AB_637945 1:500 (western blot), 1:50 (IHC) Antibody mouse anti-cav1 (monoclonal) Santa Cruz sc-53564, AB_628859 1:500 (western blot), 1:50 (IHC), 1:50 (FRIL) Antibody rabbit anti-cav1 (polyclonal) Santa Cruz sc-894 AB_2072042 1:1000 (western blot), 1:200 (IHC), 1:50 (FRIL) Antibody rabbit anti-IRTK (polyclonal) Santa Cruz sc-710, AB_631106 1:1000 (western blot) Antibody mouse anti-CAVIN-1 (monoclonal) BD Bioscience (Franklin Lakes, New Jersey) 611258, AB_398788 1:50 (FRIL) Antibody mouse anti-PDI (monoclonal) Stressgene (Farmingdale, New York) ADI-SPA-891, AB_10615355 1:1000 (western blot) Antibody mouse anti-b-actin (monoclonal) Sigma A5441, AB_476744 1:5000 (western blot) Antibody mouse anti-b-tubulin (monoclonal) Sigma T4026, AB_477577 1:1500 (western blot) Antibody rabbit anti-CAVIN-1 (polyclonal) Proteintech (Rosemont, Illinois) 18892–1-AP, AB_10596795 1:1000 (western blot), 1:100 (IHC), 1:50 (FRIL) Antibody rabbit anti-CAVIN-3 (polyclonal) Proteintech 16250–1-AP AB_2171894 1:1000 (western blot) Antibody goat anti-CAVIN-2 (polyclonal) R & D Systems (Minneapolis, Minnesota) AF5759 AB_2269901 1:200 (western blot) Antibody rabbit anti-CAVIN-4 (polyclonal) Sigma HPA020987 AB_1853080 1:400 (western blot) Continued on next page Seemann et al. eLife 2017;6:e29854.

Techniques: Mutagenesis, Staining, Muscles, Quantitation Assay