alpha-sarcoglycan Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 89
    Novocastra α sarcoglycan
    Expression of wild-type <t>α-sarcoglycan</t> in the βγδ-HEK cells promotes the localization of sarcoglycan complex to the plasma membrane. A: Western blot analysis of sarcoglycans (α-SG, β-SG, γ-SG, and δ-SG) in lysates of βγδ-HEK either mock transfected (m) or transfected with wild-type α-sarcoglycan (α). B: Western blot analysis of sarcoglycans biotinylated proteins (surface proteins) isolated by streptavidin affinity chromatography from βγδ-HEK cells either mock transfected (m) or transfected with wild-type α-sarcoglycan (α). β-Actin (β-act) and β-dystroglycan (β-DG) were used as cytoplasmic and surface protein markers, respectively. Each protein was identified by the specific antibody. A representative of two independent experiments is shown. C: Immunoprecipitation of sarcoglycans was performed on the biotinylated cell surface proteins of the βγδ-HEK cells by using the antibody specific to β-sarcoglycan. The sarcoglycan complex was sedimented only from the βγδ-HEK cells transfected with α-sarcoglycan (α) and not from the mock transfected cells (m). β-Dystroglycan (β-DG) co-sedimented with the sarcoglycan tetramer. Sarcoglycan proteins were revealed by the monoclonal specific antibodies (α-, β-, and γ-sarcoglycan) and the polyclonal antibody to δ-sarcoglycan. The arrows indicate mouse IgG heavy and light chains. Note that α-sarcoglycan protein band partially co-migrates with the IgG heavy chain band. The asterisk indicates a nonspecific protein band detected by the antibody to δ-sarcoglycan. A representative of two independent experiments is shown.
    α Sarcoglycan, supplied by Novocastra, used in various techniques. Bioz Stars score: 89/100, based on 188 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/α sarcoglycan/product/Novocastra
    Average 89 stars, based on 188 article reviews
    Price from $9.99 to $1999.99
    α sarcoglycan - by Bioz Stars, 2020-12
    89/100 stars
      Buy from Supplier

    89
    Novocastra β sarcoglycan
    In vivo cross-linking of the sarcoglycans. Mouse myotubes were chemically cross-linked with 1 mM DTSSP and immunoprecipitated by <t>anti–β-sarcoglycan</t> antibody ( A ) or anti–α- or β-dystroglycan antibody ( B ). Lane 1 , cell lysate from mouse myotubes (26–34% input). Lanes 2 and 4 , immunoprecipitated products from uncross-linked myotubes. Lanes 3 and 5 , immunoprecipitated products from equal amount of DTSSP cross-linked myotubes. The composition of the immune complex was determined by Western blots using antibodies ( 1 ° Ab ) against different sarcoglycans and dystroglycans.
    β Sarcoglycan, supplied by Novocastra, used in various techniques. Bioz Stars score: 89/100, based on 160 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/β sarcoglycan/product/Novocastra
    Average 89 stars, based on 160 article reviews
    Price from $9.99 to $1999.99
    β sarcoglycan - by Bioz Stars, 2020-12
    89/100 stars
      Buy from Supplier

    89
    Novocastra γ sarcoglycan
    Hexokinase II is also reduced in the MRL cardiac tissue. a Representative immunoblot of HKII in the heart tissues. b <t>γ-Sarcoglycan</t> loading control. c Quantification of HKII normalized to γ-sarcoglycan and B6 CD quantities (N = 12, B6 CD verses MRL CD p = 0.000, B6 HFD versus MRL HFD p = 0.001, and B6 CD versus B6 HFD p = 0.013)
    γ Sarcoglycan, supplied by Novocastra, used in various techniques. Bioz Stars score: 89/100, based on 89 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ sarcoglycan/product/Novocastra
    Average 89 stars, based on 89 article reviews
    Price from $9.99 to $1999.99
    γ sarcoglycan - by Bioz Stars, 2020-12
    89/100 stars
      Buy from Supplier

    89
    Novocastra δ sarcoglycan
    Absence of intermolecular disulfide bonds between the sarcoglycans. Cell lysate from cultured mouse myotubes was electrophoresed in 2-D diagonal gel and examined by Western blots using antibodies against different sarcoglycans. Shown in the example is the Western blot using antibodies against α-, β-, and <t>δ-sarcoglycan.</t> Note that no extra spot was observed below the diagonal line ( dashed line ).
    δ Sarcoglycan, supplied by Novocastra, used in various techniques. Bioz Stars score: 89/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/δ sarcoglycan/product/Novocastra
    Average 89 stars, based on 38 article reviews
    Price from $9.99 to $1999.99
    δ sarcoglycan - by Bioz Stars, 2020-12
    89/100 stars
      Buy from Supplier

    85
    Takeda takeda s epsilon sarcoglycan
    Absence of intermolecular disulfide bonds between the sarcoglycans. Cell lysate from cultured mouse myotubes was electrophoresed in 2-D diagonal gel and examined by Western blots using antibodies against different sarcoglycans. Shown in the example is the Western blot using antibodies against α-, β-, and <t>δ-sarcoglycan.</t> Note that no extra spot was observed below the diagonal line ( dashed line ).
    Takeda S Epsilon Sarcoglycan, supplied by Takeda, used in various techniques. Bioz Stars score: 85/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/takeda s epsilon sarcoglycan/product/Takeda
    Average 85 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    takeda s epsilon sarcoglycan - by Bioz Stars, 2020-12
    85/100 stars
      Buy from Supplier

    92
    Leica Biosystems α sarcoglycan
    Disruption of dystrophin expression in the skeletal muscle of DMD KO rabbits. (A-C) Immunofluorescence staining of muscle sections from WT and DMD KO rabbits with mouse monoclonal antibodies against dystrophin (A), glycosylated α-dystroglycan (B) and <t>α-sarcoglycan</t> (C). Nuclei were stained by DAPI. Scale bar: 100 µm.
    α Sarcoglycan, supplied by Leica Biosystems, used in various techniques. Bioz Stars score: 92/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/α sarcoglycan/product/Leica Biosystems
    Average 92 stars, based on 27 article reviews
    Price from $9.99 to $1999.99
    α sarcoglycan - by Bioz Stars, 2020-12
    92/100 stars
      Buy from Supplier

    90
    Abcam δ sarcoglycan
    Tsp expression in muscle of Drosophila rescues MD due to deletion of the <t>δ-sarcoglycan-like</t> gene (Sgcd 840 ). ( A ) Fly survival was compared over a period of 40 days. p
    δ Sarcoglycan, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/δ sarcoglycan/product/Abcam
    Average 90 stars, based on 31 article reviews
    Price from $9.99 to $1999.99
    δ sarcoglycan - by Bioz Stars, 2020-12
    90/100 stars
      Buy from Supplier

    89
    Santa Cruz Biotechnology δ sarcoglycan
    Generation of <t>dystrophin/δ-sarcoglycan</t> double mutant mice. ( A ) The breeding scheme. ( B ) Representative PCR genotyping results. Bkg, background; δSG KO, δ-sarcoglycan deficient mice; δ-Dko, δ-sarcoglycan/dystrophin
    δ Sarcoglycan, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 89/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/δ sarcoglycan/product/Santa Cruz Biotechnology
    Average 89 stars, based on 29 article reviews
    Price from $9.99 to $1999.99
    δ sarcoglycan - by Bioz Stars, 2020-12
    89/100 stars
      Buy from Supplier

    94
    Developmental Studies Hybridoma Bank α sarcoglycan
    Rac1 regulated exercise‐induced GLUT4 translocation A , representative images showing cryosections of tibialis anterior muscle (cross sections) from basal and exercise‐stimulated Rac1 mKO and WT littermate mice stained with antibodies against GLUT4 (green) and <t>α‐sarcoglycan</t> (red). Scale bar = 50 μm. B , illustration of the quantification approach. Red arrows indicate the locations of the lines across and perpendicular to the plasma membrane of which 5 μm were analysed and quantified. C , quantification of the intensity of plasma membrane GLUT4 relative to α‐sarcoglycan in the basal and exercise‐stimulated (65% of individual maximal running speed, 20 min) state of WT and Rac1 mKO tibialis anterior muscle ( n = 4–6). D , 2‐DG uptake in basal and exercise‐stimulated (65% of individual maximum running speed, 20 min) tibialis anterior muscle ( n = 5 or 6). E , correlation between plasma membrane GLUT4 (GLUT4/α‐sarcoglycan) and 2‐DG uptake in basal and exercise‐stimulated tibialis anterior muscle. Included are only samples analysed for both parameters in the same muscle ( n = 4 or 5). Significant difference between basal and exercise‐stimulated intensity of GLUT4/α‐sarcoglycan or 2‐DG uptake is indicated: * P
    α Sarcoglycan, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 94/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/α sarcoglycan/product/Developmental Studies Hybridoma Bank
    Average 94 stars, based on 28 article reviews
    Price from $9.99 to $1999.99
    α sarcoglycan - by Bioz Stars, 2020-12
    94/100 stars
      Buy from Supplier

    89
    Leica Microsystems β sarcoglycan
    Differences in α-dystrobrevin splice variants between cardiac and skeletal muscle DAPC. ( A ) Western blot analysis of α-dystrobrevins in mouse cardiac (C) and skeletal (S) muscle total protein lysates, DYS-IPs and IgG-IPs. α3-dystrobrevin associates with dystrophin in the heart. Fold differences in α-dystrobrevin abundance in cardiac vs. skeletal muscle DYS-IPs relative to dystrophin are shown (averages ±SD, N = 3). ( B ) Immunolabeling of wild type cardiac sections for α1 and α2-dystrobrevins. Scale bar: 50 µm. ( C ) Immunolabeling of mdx cardiac tissue section for <t>β-sarcoglycan.</t> Scale bar: 50 µm. ( D ) Western blot analysis of α-dystrobrevins in heart protein lysates from wild type (WT) and mdx mice. Fold differences in α-dystrobrevin abundance in WT vs. mdx cardiac lysates relative to GAPDH are shown (averages ±SD, N = 3) ( E ) Western blot analysis of α-dystrobrevins in DYS-IPs from human (H) and mouse (M) cardiac samples. Additional α-dystrobrevin isoforms (arrow heads) are detected in human cardiac lysates and DYS-IP.
    β Sarcoglycan, supplied by Leica Microsystems, used in various techniques. Bioz Stars score: 89/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/β sarcoglycan/product/Leica Microsystems
    Average 89 stars, based on 28 article reviews
    Price from $9.99 to $1999.99
    β sarcoglycan - by Bioz Stars, 2020-12
    89/100 stars
      Buy from Supplier

    89
    Leica Microsystems α sarcoglycan
    Differences in α-dystrobrevin splice variants between cardiac and skeletal muscle DAPC. ( A ) Western blot analysis of α-dystrobrevins in mouse cardiac (C) and skeletal (S) muscle total protein lysates, DYS-IPs and IgG-IPs. α3-dystrobrevin associates with dystrophin in the heart. Fold differences in α-dystrobrevin abundance in cardiac vs. skeletal muscle DYS-IPs relative to dystrophin are shown (averages ±SD, N = 3). ( B ) Immunolabeling of wild type cardiac sections for α1 and α2-dystrobrevins. Scale bar: 50 µm. ( C ) Immunolabeling of mdx cardiac tissue section for <t>β-sarcoglycan.</t> Scale bar: 50 µm. ( D ) Western blot analysis of α-dystrobrevins in heart protein lysates from wild type (WT) and mdx mice. Fold differences in α-dystrobrevin abundance in WT vs. mdx cardiac lysates relative to GAPDH are shown (averages ±SD, N = 3) ( E ) Western blot analysis of α-dystrobrevins in DYS-IPs from human (H) and mouse (M) cardiac samples. Additional α-dystrobrevin isoforms (arrow heads) are detected in human cardiac lysates and DYS-IP.
    α Sarcoglycan, supplied by Leica Microsystems, used in various techniques. Bioz Stars score: 89/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/α sarcoglycan/product/Leica Microsystems
    Average 89 stars, based on 23 article reviews
    Price from $9.99 to $1999.99
    α sarcoglycan - by Bioz Stars, 2020-12
    89/100 stars
      Buy from Supplier

    93
    Novocastra mouse monoclonal anti α sarcoglycan
    Dependence of ecto-nucleotidase activity of <t>α-sarcoglycan-expressing</t> HEK-293 cells on the bivalent cation concentrations ( A ) ATP-hydrolysing activity was measured in stably transfected cells in the presence of 4 mM Mg 2+ or 2 mM Ca 2+ or both (4 mM Mg 2+ and 2 mM Ca 2+ ). ( B ) ATP-hydrolysing activity of α-sarcoglycan stably transfected HEK-293 cells was measured either in the presence of 4 mM Mg 2+ and the indicated concentrations of Ca 2+ (○) or in the presence of 2 mM Ca 2+ and the indicated concentrations of Mg 2+ (•). The plot shows the values after the subtraction of the empty vector-transfected activity. Results are from four experiments performed in triplicate. ** P
    Mouse Monoclonal Anti α Sarcoglycan, supplied by Novocastra, used in various techniques. Bioz Stars score: 93/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse monoclonal anti α sarcoglycan/product/Novocastra
    Average 93 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    mouse monoclonal anti α sarcoglycan - by Bioz Stars, 2020-12
    93/100 stars
      Buy from Supplier

    89
    Santa Cruz Biotechnology β sarcoglycan
    Dependence of ecto-nucleotidase activity of <t>α-sarcoglycan-expressing</t> HEK-293 cells on the bivalent cation concentrations ( A ) ATP-hydrolysing activity was measured in stably transfected cells in the presence of 4 mM Mg 2+ or 2 mM Ca 2+ or both (4 mM Mg 2+ and 2 mM Ca 2+ ). ( B ) ATP-hydrolysing activity of α-sarcoglycan stably transfected HEK-293 cells was measured either in the presence of 4 mM Mg 2+ and the indicated concentrations of Ca 2+ (○) or in the presence of 2 mM Ca 2+ and the indicated concentrations of Mg 2+ (•). The plot shows the values after the subtraction of the empty vector-transfected activity. Results are from four experiments performed in triplicate. ** P
    β Sarcoglycan, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 89/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/β sarcoglycan/product/Santa Cruz Biotechnology
    Average 89 stars, based on 16 article reviews
    Price from $9.99 to $1999.99
    β sarcoglycan - by Bioz Stars, 2020-12
    89/100 stars
      Buy from Supplier

    92
    Leica Biosystems γ sarcoglycan
    Dependence of ecto-nucleotidase activity of <t>α-sarcoglycan-expressing</t> HEK-293 cells on the bivalent cation concentrations ( A ) ATP-hydrolysing activity was measured in stably transfected cells in the presence of 4 mM Mg 2+ or 2 mM Ca 2+ or both (4 mM Mg 2+ and 2 mM Ca 2+ ). ( B ) ATP-hydrolysing activity of α-sarcoglycan stably transfected HEK-293 cells was measured either in the presence of 4 mM Mg 2+ and the indicated concentrations of Ca 2+ (○) or in the presence of 2 mM Ca 2+ and the indicated concentrations of Mg 2+ (•). The plot shows the values after the subtraction of the empty vector-transfected activity. Results are from four experiments performed in triplicate. ** P
    γ Sarcoglycan, supplied by Leica Biosystems, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ sarcoglycan/product/Leica Biosystems
    Average 92 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    γ sarcoglycan - by Bioz Stars, 2020-12
    92/100 stars
      Buy from Supplier

    89
    Leica Microsystems γ sarcoglycan
    Dependence of ecto-nucleotidase activity of <t>α-sarcoglycan-expressing</t> HEK-293 cells on the bivalent cation concentrations ( A ) ATP-hydrolysing activity was measured in stably transfected cells in the presence of 4 mM Mg 2+ or 2 mM Ca 2+ or both (4 mM Mg 2+ and 2 mM Ca 2+ ). ( B ) ATP-hydrolysing activity of α-sarcoglycan stably transfected HEK-293 cells was measured either in the presence of 4 mM Mg 2+ and the indicated concentrations of Ca 2+ (○) or in the presence of 2 mM Ca 2+ and the indicated concentrations of Mg 2+ (•). The plot shows the values after the subtraction of the empty vector-transfected activity. Results are from four experiments performed in triplicate. ** P
    γ Sarcoglycan, supplied by Leica Microsystems, used in various techniques. Bioz Stars score: 89/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ sarcoglycan/product/Leica Microsystems
    Average 89 stars, based on 19 article reviews
    Price from $9.99 to $1999.99
    γ sarcoglycan - by Bioz Stars, 2020-12
    89/100 stars
      Buy from Supplier

    89
    Santa Cruz Biotechnology γ sarcoglycan
    The expression analyses of α-, β-, <t>γ-sarcoglycan</t> and dysferlin protein in the muscles of patient by Western blot. The total proteins extracted from skeletal muscles of normal person (lane 1), relative normal muscle biopsies of patient IV-7 (lane 2), and dystrophic muscle biopsies of patient IV-7 (lane 3).
    γ Sarcoglycan, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 89/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ sarcoglycan/product/Santa Cruz Biotechnology
    Average 89 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    γ sarcoglycan - by Bioz Stars, 2020-12
    89/100 stars
      Buy from Supplier

    92
    Abnova rabbit polyclonal anti γ sarcoglycan
    SGCG reading frame correction in urine-derived cells (UDCs). UDCs from a normal control subject and an LGMD 2C patient with a deletion of exon 6 (ex6del) were reprogrammed into a myogenic lineage. ( A ) <t>γ-Sarcoglycan</t> protein (green) was detected by IFM in the reprogrammed normal control myotubes but not in reprogrammed ex6del cells. α-Actinin, red; nuclei, blue. ( B ) RT-PCR analysis demonstrated reading frame–corrected Mini-Gamma transcript expression (red arrowhead). ( C ) Representative IFM images showed the restoration of γ-sarcoglycan protein expression in cells treated with Mini-Gamma vivo-PMOs. ( D ) Significant increase in γ-sarcoglycan protein fluorescence was observed after treatment with vivo-PMOs ( n = 5) as compared with nontargeting control vivo-PMOs ( n = 5). A minimum of 3 independent fields were analyzed for each sample. ( E ) To assess membrane stability in response to vivo-PMO treatment, reprogrammed cells were challenged with hypo-osmotic shock and membrane leak was monitored by release of creatine kinase (CK). Vivo-PMO treatment significantly decreased the relative amount of CK release consistent with increased membrane stability. Data represent the percent of CK released relative to the total CK from 4 independent experiments ( n = 3–4, for each). Data are presented as the mean CK released in cells treated with exon-skipping vivo-PMOs relative to the mean in cells treated with a nontargeting vivo-PMO. ( F ) Model depicting the increased membrane stability that resulted from vivo-PMO–mediated reading frame correction of an SGCG frameshift mutation. * P
    Rabbit Polyclonal Anti γ Sarcoglycan, supplied by Abnova, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal anti γ sarcoglycan/product/Abnova
    Average 92 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    rabbit polyclonal anti γ sarcoglycan - by Bioz Stars, 2020-12
    92/100 stars
      Buy from Supplier

    Image Search Results


    Expression of wild-type α-sarcoglycan in the βγδ-HEK cells promotes the localization of sarcoglycan complex to the plasma membrane. A: Western blot analysis of sarcoglycans (α-SG, β-SG, γ-SG, and δ-SG) in lysates of βγδ-HEK either mock transfected (m) or transfected with wild-type α-sarcoglycan (α). B: Western blot analysis of sarcoglycans biotinylated proteins (surface proteins) isolated by streptavidin affinity chromatography from βγδ-HEK cells either mock transfected (m) or transfected with wild-type α-sarcoglycan (α). β-Actin (β-act) and β-dystroglycan (β-DG) were used as cytoplasmic and surface protein markers, respectively. Each protein was identified by the specific antibody. A representative of two independent experiments is shown. C: Immunoprecipitation of sarcoglycans was performed on the biotinylated cell surface proteins of the βγδ-HEK cells by using the antibody specific to β-sarcoglycan. The sarcoglycan complex was sedimented only from the βγδ-HEK cells transfected with α-sarcoglycan (α) and not from the mock transfected cells (m). β-Dystroglycan (β-DG) co-sedimented with the sarcoglycan tetramer. Sarcoglycan proteins were revealed by the monoclonal specific antibodies (α-, β-, and γ-sarcoglycan) and the polyclonal antibody to δ-sarcoglycan. The arrows indicate mouse IgG heavy and light chains. Note that α-sarcoglycan protein band partially co-migrates with the IgG heavy chain band. The asterisk indicates a nonspecific protein band detected by the antibody to δ-sarcoglycan. A representative of two independent experiments is shown.

    Journal: The American Journal of Pathology

    Article Title: Inhibition of Proteasome Activity Promotes the Correct Localization of Disease-Causing ?-Sarcoglycan Mutants in HEK-293 Cells Constitutively Expressing ?-, ?-, and ?-Sarcoglycan

    doi: 10.2353/ajpath.2008.071146

    Figure Lengend Snippet: Expression of wild-type α-sarcoglycan in the βγδ-HEK cells promotes the localization of sarcoglycan complex to the plasma membrane. A: Western blot analysis of sarcoglycans (α-SG, β-SG, γ-SG, and δ-SG) in lysates of βγδ-HEK either mock transfected (m) or transfected with wild-type α-sarcoglycan (α). B: Western blot analysis of sarcoglycans biotinylated proteins (surface proteins) isolated by streptavidin affinity chromatography from βγδ-HEK cells either mock transfected (m) or transfected with wild-type α-sarcoglycan (α). β-Actin (β-act) and β-dystroglycan (β-DG) were used as cytoplasmic and surface protein markers, respectively. Each protein was identified by the specific antibody. A representative of two independent experiments is shown. C: Immunoprecipitation of sarcoglycans was performed on the biotinylated cell surface proteins of the βγδ-HEK cells by using the antibody specific to β-sarcoglycan. The sarcoglycan complex was sedimented only from the βγδ-HEK cells transfected with α-sarcoglycan (α) and not from the mock transfected cells (m). β-Dystroglycan (β-DG) co-sedimented with the sarcoglycan tetramer. Sarcoglycan proteins were revealed by the monoclonal specific antibodies (α-, β-, and γ-sarcoglycan) and the polyclonal antibody to δ-sarcoglycan. The arrows indicate mouse IgG heavy and light chains. Note that α-sarcoglycan protein band partially co-migrates with the IgG heavy chain band. The asterisk indicates a nonspecific protein band detected by the antibody to δ-sarcoglycan. A representative of two independent experiments is shown.

    Article Snippet: Mouse monoclonal antibodies specific for α-sarcoglycan (NCL-a-SARC), β-sarcoglycan (NCL-b-SARC), γ-sarcoglycan (NCL-g-SARC), were from Novocastra (Newcastle upon Tyne, UK).

    Techniques: Expressing, Western Blot, Transfection, Isolation, Affinity Chromatography, Activated Clotting Time Assay, Immunoprecipitation

    Treatment with proteasome inhibitor MG132 promotes the cell membrane localization of sarcoglycan complex. βγδ-HEK cells were transiently transfected either with wild-type α-sarcoglycan (wt α-SG) or singly with R77C, D97G, R98H, P228Q, and V247M α-sarcoglycan mutants. Transfected cells were then incubated with (+) or without (−) 10 μmol/L MG132 proteasome inhibitor as indicated in Materials and Methods. Cells surface proteins were biotinylated, purified by streptavidin affinity chromatography and finally analyzed by Western blot by using specific antibodies to α-, β-, γ-, and δ-sarcoglycan (α-, β-, γ-, and δ-SG) and β-dystroglycan (β-DG). A representative out of three independent experiments is shown.

    Journal: The American Journal of Pathology

    Article Title: Inhibition of Proteasome Activity Promotes the Correct Localization of Disease-Causing ?-Sarcoglycan Mutants in HEK-293 Cells Constitutively Expressing ?-, ?-, and ?-Sarcoglycan

    doi: 10.2353/ajpath.2008.071146

    Figure Lengend Snippet: Treatment with proteasome inhibitor MG132 promotes the cell membrane localization of sarcoglycan complex. βγδ-HEK cells were transiently transfected either with wild-type α-sarcoglycan (wt α-SG) or singly with R77C, D97G, R98H, P228Q, and V247M α-sarcoglycan mutants. Transfected cells were then incubated with (+) or without (−) 10 μmol/L MG132 proteasome inhibitor as indicated in Materials and Methods. Cells surface proteins were biotinylated, purified by streptavidin affinity chromatography and finally analyzed by Western blot by using specific antibodies to α-, β-, γ-, and δ-sarcoglycan (α-, β-, γ-, and δ-SG) and β-dystroglycan (β-DG). A representative out of three independent experiments is shown.

    Article Snippet: Mouse monoclonal antibodies specific for α-sarcoglycan (NCL-a-SARC), β-sarcoglycan (NCL-b-SARC), γ-sarcoglycan (NCL-g-SARC), were from Novocastra (Newcastle upon Tyne, UK).

    Techniques: Transfection, Incubation, Purification, Affinity Chromatography, Western Blot

    Confocal immunofluorescence analysis confirms the cell membrane localization of disease-causing α-sarcoglycan mutants after MG132 treatment. βγδ-HEK cells were transiently transfected either with wild-type α-sarcoglycan (wt α-SG) or singly with R77C, D97G, R98H, P228Q, and V247M α-sarcoglycan mutants. Transfected cells were treated with proteasome inhibitor MG132 (10 μmol/L, for 8 hours) or with vehicle. Nonpermeabilized cells were decorated with the antibody specific for α-sarcoglycan. The phase contrast images of transfected cells were merged with the images of the same cells stained with the antibody. Images are representative of three independent experiments.

    Journal: The American Journal of Pathology

    Article Title: Inhibition of Proteasome Activity Promotes the Correct Localization of Disease-Causing ?-Sarcoglycan Mutants in HEK-293 Cells Constitutively Expressing ?-, ?-, and ?-Sarcoglycan

    doi: 10.2353/ajpath.2008.071146

    Figure Lengend Snippet: Confocal immunofluorescence analysis confirms the cell membrane localization of disease-causing α-sarcoglycan mutants after MG132 treatment. βγδ-HEK cells were transiently transfected either with wild-type α-sarcoglycan (wt α-SG) or singly with R77C, D97G, R98H, P228Q, and V247M α-sarcoglycan mutants. Transfected cells were treated with proteasome inhibitor MG132 (10 μmol/L, for 8 hours) or with vehicle. Nonpermeabilized cells were decorated with the antibody specific for α-sarcoglycan. The phase contrast images of transfected cells were merged with the images of the same cells stained with the antibody. Images are representative of three independent experiments.

    Article Snippet: Mouse monoclonal antibodies specific for α-sarcoglycan (NCL-a-SARC), β-sarcoglycan (NCL-b-SARC), γ-sarcoglycan (NCL-g-SARC), were from Novocastra (Newcastle upon Tyne, UK).

    Techniques: Immunofluorescence, Transfection, Staining

    Expression of α-sarcoglycan mutants in βγδ-HEK cells: effects of proteasome inhibitors. A: Densitometric analysis of the expression level of α-sarcoglycan mutants in lysates of βγδ-HEK cells transfected with either wild-type α-sarcoglycan (wt-αSG) or α-sarcoglycan bearing the indicated disease-causing missense mutations (three to six experiments). The relative protein expression refers to that of wild-type α-sarcoglycan transfected in the βγδ-HEK cells normalized to the content of β-actin. B: Western blot analysis of the immunoprecipitated proteins from βγδ-HEK cells transfected with the indicated constructs and treated for 8 hours with 20 μmol/L lactacystin (+) or with vehicle (−). Wild-type or α-sarcoglycan mutant proteins were immunoprecipitated with the monoclonal antibody to α-sarcoglycan and probed with the polyclonal antibody to α-sarcoglycan (lower blots, arrowhead indicates a non specific band). Thereafter, antibodies were stripped from the filter, which was then probed with an antibody to ubiquitin (upper blots, the arrows indicate mouse IgG heavy chains and traces of α-sarcoglycan remained after stripping). C: Western blot and densitometric analyses of lysates of βγδ-HEK cells transfected with either wild-type α-sarcoglycan or α-sarcoglycan mutants (three to six experiments). Either the proteasome inhibitor MG132 (10 μmol/L for 8 hours) (+) or the sole vehicle (−) were applied to transfected cells. β-Actin was used as an internal marker to normalize cell protein content. The protein indicated by an arrow (ng), recognized by the α-sarcoglycan antibody, was identified as the nonglycosylated form of α-sarcoglycan. In the densitometric analysis, the relative expression refers to that of wild-type α-sarcoglycan expressed in the βγδ-HEK untreated cells. * P

    Journal: The American Journal of Pathology

    Article Title: Inhibition of Proteasome Activity Promotes the Correct Localization of Disease-Causing ?-Sarcoglycan Mutants in HEK-293 Cells Constitutively Expressing ?-, ?-, and ?-Sarcoglycan

    doi: 10.2353/ajpath.2008.071146

    Figure Lengend Snippet: Expression of α-sarcoglycan mutants in βγδ-HEK cells: effects of proteasome inhibitors. A: Densitometric analysis of the expression level of α-sarcoglycan mutants in lysates of βγδ-HEK cells transfected with either wild-type α-sarcoglycan (wt-αSG) or α-sarcoglycan bearing the indicated disease-causing missense mutations (three to six experiments). The relative protein expression refers to that of wild-type α-sarcoglycan transfected in the βγδ-HEK cells normalized to the content of β-actin. B: Western blot analysis of the immunoprecipitated proteins from βγδ-HEK cells transfected with the indicated constructs and treated for 8 hours with 20 μmol/L lactacystin (+) or with vehicle (−). Wild-type or α-sarcoglycan mutant proteins were immunoprecipitated with the monoclonal antibody to α-sarcoglycan and probed with the polyclonal antibody to α-sarcoglycan (lower blots, arrowhead indicates a non specific band). Thereafter, antibodies were stripped from the filter, which was then probed with an antibody to ubiquitin (upper blots, the arrows indicate mouse IgG heavy chains and traces of α-sarcoglycan remained after stripping). C: Western blot and densitometric analyses of lysates of βγδ-HEK cells transfected with either wild-type α-sarcoglycan or α-sarcoglycan mutants (three to six experiments). Either the proteasome inhibitor MG132 (10 μmol/L for 8 hours) (+) or the sole vehicle (−) were applied to transfected cells. β-Actin was used as an internal marker to normalize cell protein content. The protein indicated by an arrow (ng), recognized by the α-sarcoglycan antibody, was identified as the nonglycosylated form of α-sarcoglycan. In the densitometric analysis, the relative expression refers to that of wild-type α-sarcoglycan expressed in the βγδ-HEK untreated cells. * P

    Article Snippet: Mouse monoclonal antibodies specific for α-sarcoglycan (NCL-a-SARC), β-sarcoglycan (NCL-b-SARC), γ-sarcoglycan (NCL-g-SARC), were from Novocastra (Newcastle upon Tyne, UK).

    Techniques: Expressing, Transfection, Western Blot, Immunoprecipitation, Construct, Mutagenesis, Stripping Membranes, Marker

    Treatment with the FDA-approved proteasome inhibitor Velcade causes the dose-dependent increased expression of V247M α-sarcoglycan mutant. A: Survival of untransfected HEK-293 cells after treatment for 4 (empty bars), 8 (gray bars), or 24 (black bars) hours with increasing concentrations of Velcade (5 nmol/L to 50 μmol/L). B: βγδ-HEK cells transfected with V247M α-sarcoglycan were treated with increasing concentration of Velcade for 8 hours. βγδ-HEK cells transfected with wild-type α-sarcoglycan (wt-αSG) were used as control. Western blot of cell lysates were probed with antibodies specific for α-sarcoglycan and β-actin, used as an internal marker to normalize cell protein content. The protein indicated by an asterisk , sensitive to the α-sarcoglycan antibody, was identified, by probing the lysates with PNGase F, as the nonglycosylated form of α-sarcoglycan. The α-sarcoglycan expression level was determined by densitometric analysis performed in at least three independent experiments (a representative one is shown).

    Journal: The American Journal of Pathology

    Article Title: Inhibition of Proteasome Activity Promotes the Correct Localization of Disease-Causing ?-Sarcoglycan Mutants in HEK-293 Cells Constitutively Expressing ?-, ?-, and ?-Sarcoglycan

    doi: 10.2353/ajpath.2008.071146

    Figure Lengend Snippet: Treatment with the FDA-approved proteasome inhibitor Velcade causes the dose-dependent increased expression of V247M α-sarcoglycan mutant. A: Survival of untransfected HEK-293 cells after treatment for 4 (empty bars), 8 (gray bars), or 24 (black bars) hours with increasing concentrations of Velcade (5 nmol/L to 50 μmol/L). B: βγδ-HEK cells transfected with V247M α-sarcoglycan were treated with increasing concentration of Velcade for 8 hours. βγδ-HEK cells transfected with wild-type α-sarcoglycan (wt-αSG) were used as control. Western blot of cell lysates were probed with antibodies specific for α-sarcoglycan and β-actin, used as an internal marker to normalize cell protein content. The protein indicated by an asterisk , sensitive to the α-sarcoglycan antibody, was identified, by probing the lysates with PNGase F, as the nonglycosylated form of α-sarcoglycan. The α-sarcoglycan expression level was determined by densitometric analysis performed in at least three independent experiments (a representative one is shown).

    Article Snippet: Mouse monoclonal antibodies specific for α-sarcoglycan (NCL-a-SARC), β-sarcoglycan (NCL-b-SARC), γ-sarcoglycan (NCL-g-SARC), were from Novocastra (Newcastle upon Tyne, UK).

    Techniques: Expressing, Mutagenesis, Transfection, Concentration Assay, Western Blot, Marker

    Treatment with proteasome inhibitor MG132 of βγδ-HEK cells transfected with V247M α-sarcoglycan mutant promotes the organization of a functional sarcoglycan complex at the cell membrane. βγδ-HEK cells were transiently transfected with V247M α-sarcoglycan mutant and incubated with 10 μmol/L MG132 proteasome inhibitor as indicated in Materials and Methods. Immunoprecipitation of sarcoglycans was performed on the biotinylated cell surface proteins by using the antibody specific to β-sarcoglycan. The four sarcoglycans form a functional complex at the cell membrane as they co-sedimented with β-dystroglycan (β-DG). As expected the intracellular protein β-actin was not immunoprecipitated. Sarcoglycan proteins were revealed by the monoclonal specific antibodies (α-, β-, and γ-sarcoglycan, α-SG, β-SG, and γ-SG, respectively) and the polyclonal antibody to α- and δ-sarcoglycan (δ-SG). The arrows indicate mouse IgG heavy and light chains. Note that the α-sarcoglycan protein band partially co-migrates with the IgG heavy chain band. To better demonstrate the presence of V247M α-sarcoglycan mutant in the immunoprecipitated complex, the filter probed with the monoclonal antibody to α-sarcoglycan (m) was stripped and then probed with the polyclonal antibody (p); the reaction was revealed by ECL. The asterisk indicates a nonspecific protein band detected by the antibody to δ-sarcoglycan.

    Journal: The American Journal of Pathology

    Article Title: Inhibition of Proteasome Activity Promotes the Correct Localization of Disease-Causing ?-Sarcoglycan Mutants in HEK-293 Cells Constitutively Expressing ?-, ?-, and ?-Sarcoglycan

    doi: 10.2353/ajpath.2008.071146

    Figure Lengend Snippet: Treatment with proteasome inhibitor MG132 of βγδ-HEK cells transfected with V247M α-sarcoglycan mutant promotes the organization of a functional sarcoglycan complex at the cell membrane. βγδ-HEK cells were transiently transfected with V247M α-sarcoglycan mutant and incubated with 10 μmol/L MG132 proteasome inhibitor as indicated in Materials and Methods. Immunoprecipitation of sarcoglycans was performed on the biotinylated cell surface proteins by using the antibody specific to β-sarcoglycan. The four sarcoglycans form a functional complex at the cell membrane as they co-sedimented with β-dystroglycan (β-DG). As expected the intracellular protein β-actin was not immunoprecipitated. Sarcoglycan proteins were revealed by the monoclonal specific antibodies (α-, β-, and γ-sarcoglycan, α-SG, β-SG, and γ-SG, respectively) and the polyclonal antibody to α- and δ-sarcoglycan (δ-SG). The arrows indicate mouse IgG heavy and light chains. Note that the α-sarcoglycan protein band partially co-migrates with the IgG heavy chain band. To better demonstrate the presence of V247M α-sarcoglycan mutant in the immunoprecipitated complex, the filter probed with the monoclonal antibody to α-sarcoglycan (m) was stripped and then probed with the polyclonal antibody (p); the reaction was revealed by ECL. The asterisk indicates a nonspecific protein band detected by the antibody to δ-sarcoglycan.

    Article Snippet: Mouse monoclonal antibodies specific for α-sarcoglycan (NCL-a-SARC), β-sarcoglycan (NCL-b-SARC), γ-sarcoglycan (NCL-g-SARC), were from Novocastra (Newcastle upon Tyne, UK).

    Techniques: Transfection, Mutagenesis, Functional Assay, Incubation, Immunoprecipitation

    Compound panel showing immunostaining of longitudinal sections of skeletal muscle fibres of patients with sensitive-motor polyneuropathy. The sections were immunolabelled with antibodies against α-sarcoglycan (A), β-sarcoglycan (B), γ-sarcoglycan

    Journal: Journal of Anatomy

    Article Title: Costameric proteins in human skeletal muscle during muscular inactivity

    doi: 10.1111/j.1469-7580.2008.00921.x

    Figure Lengend Snippet: Compound panel showing immunostaining of longitudinal sections of skeletal muscle fibres of patients with sensitive-motor polyneuropathy. The sections were immunolabelled with antibodies against α-sarcoglycan (A), β-sarcoglycan (B), γ-sarcoglycan

    Article Snippet: The following primary antibodies were used: anti-α-sarcoglycan diluted 1:100, anti-β-sarcoglycan diluted 1:200, anti-γ-sarcoglycan diluted 1:100, anti-δ-sarcoglycan diluted 1:50, and anti-dystrophin diluted 1:20 (all from Novocastra Laboratories, Newcastle Upon Tyne, Uk); anti-agrin diluted 1:100 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); anti-vinculin diluted 1:100, and anti-talin diluted 1:100 (both from Sigma Chemicals, St. Louis, MO, USA); anti-α7B-integrin diluted 1:50, anti-β1D-integrin diluted 1:50, and anti-α7A-integrin diluted 1:100 (synthetic peptides from the COOH terminal region; kindly provided by the laboratory of Professor Tarone, University of Turin).

    Techniques: Immunostaining

    Compound panel showing immunohistochemical findings in normal human skeletal muscle. Skeletal muscle fibres were immunolabelled with antibodies against α-sarcoglycan (A), β-sarcoglycan (B), γ-sarcoglycan (C), δ-sarcoglycan

    Journal: Journal of Anatomy

    Article Title: Costameric proteins in human skeletal muscle during muscular inactivity

    doi: 10.1111/j.1469-7580.2008.00921.x

    Figure Lengend Snippet: Compound panel showing immunohistochemical findings in normal human skeletal muscle. Skeletal muscle fibres were immunolabelled with antibodies against α-sarcoglycan (A), β-sarcoglycan (B), γ-sarcoglycan (C), δ-sarcoglycan

    Article Snippet: The following primary antibodies were used: anti-α-sarcoglycan diluted 1:100, anti-β-sarcoglycan diluted 1:200, anti-γ-sarcoglycan diluted 1:100, anti-δ-sarcoglycan diluted 1:50, and anti-dystrophin diluted 1:20 (all from Novocastra Laboratories, Newcastle Upon Tyne, Uk); anti-agrin diluted 1:100 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); anti-vinculin diluted 1:100, and anti-talin diluted 1:100 (both from Sigma Chemicals, St. Louis, MO, USA); anti-α7B-integrin diluted 1:50, anti-β1D-integrin diluted 1:50, and anti-α7A-integrin diluted 1:100 (synthetic peptides from the COOH terminal region; kindly provided by the laboratory of Professor Tarone, University of Turin).

    Techniques: Immunohistochemistry

    Cross-sectional profiles of regenerating fibres. Three serial frozen cross sections of a biopsy obtained from regenerating vastus lateralis skeletal muscle 7 days after injury induced by electrical stimulation-elicited eccentric contractions (right). Three serial cross sections from the control (uninjured) leg of the same individual are shown (left) for comparison. The sections have been stained with alpha-sarcoglycan, beta-dystroglycan or dystrophin to label the sarcolemma, along with a basement membrane protein (laminin or collagen IV) and a myogenic marker (desmin or neonatal/embryonic myosin; MHCn/e). Each column of images contains single channel and combined images for each staining. In the injured muscle, dystrophin staining is completely absent in several fibres, while the basement membrane (laminin) is preserved. MHCn/e staining is evident in some small dystrophin-negative fibres. A similar pattern is evident from the alpha-sarcoglycan and beta-dystroglycan staining, with negative fibres, together with desmin+ cells, contained within a preserved basement membrane (collagen IV). Asterisk indicates some of the necrotic fibres. Note the different profiles of the injured fibres, such as varying fibre size, and infiltrating cells either confined to the fibre periphery or dispersed throughout the fibre. Scale bar, 100 μm

    Journal: Skeletal Muscle

    Article Title: The breaking and making of healthy adult human skeletal muscle in vivo

    doi: 10.1186/s13395-017-0142-x

    Figure Lengend Snippet: Cross-sectional profiles of regenerating fibres. Three serial frozen cross sections of a biopsy obtained from regenerating vastus lateralis skeletal muscle 7 days after injury induced by electrical stimulation-elicited eccentric contractions (right). Three serial cross sections from the control (uninjured) leg of the same individual are shown (left) for comparison. The sections have been stained with alpha-sarcoglycan, beta-dystroglycan or dystrophin to label the sarcolemma, along with a basement membrane protein (laminin or collagen IV) and a myogenic marker (desmin or neonatal/embryonic myosin; MHCn/e). Each column of images contains single channel and combined images for each staining. In the injured muscle, dystrophin staining is completely absent in several fibres, while the basement membrane (laminin) is preserved. MHCn/e staining is evident in some small dystrophin-negative fibres. A similar pattern is evident from the alpha-sarcoglycan and beta-dystroglycan staining, with negative fibres, together with desmin+ cells, contained within a preserved basement membrane (collagen IV). Asterisk indicates some of the necrotic fibres. Note the different profiles of the injured fibres, such as varying fibre size, and infiltrating cells either confined to the fibre periphery or dispersed throughout the fibre. Scale bar, 100 μm

    Article Snippet: Sections were stained with various combinations of antibodies against laminin, CD56, desmin and embryonic myosin (F1.652; Developmental Studies Hybridoma Bank); neonatal myosin (NCL-MHCn; Novocastra, Leica Microsystems A/S, Ballerup, Denmark); alpha-sarcoglycan (NCL-L-a-SARC, Novocastra); beta-dystroglycan (NCL-L-a-SARC, Novocastra); myogenin (F5d, Developmental Studies Hybridoma Bank); nestin, CD68, collagen IV and dystrophin (cat. no. D8168, Sigma-Aldrich Denmark A/S, Copenhagen, Denmark); myosin type I (BA.D5, Developmental Studies Hybridoma Bank) and myosin type II (A4.74, Developmental Studies Hybridoma Bank).

    Techniques: Staining, Marker

    Hexokinase II is also reduced in the MRL cardiac tissue. a Representative immunoblot of HKII in the heart tissues. b γ-Sarcoglycan loading control. c Quantification of HKII normalized to γ-sarcoglycan and B6 CD quantities (N = 12, B6 CD verses MRL CD p = 0.000, B6 HFD versus MRL HFD p = 0.001, and B6 CD versus B6 HFD p = 0.013)

    Journal: Cardiovascular Diabetology

    Article Title: Successful metabolic adaptations leading to the prevention of high fat diet-induced murine cardiac remodeling

    doi: 10.1186/s12933-015-0286-0

    Figure Lengend Snippet: Hexokinase II is also reduced in the MRL cardiac tissue. a Representative immunoblot of HKII in the heart tissues. b γ-Sarcoglycan loading control. c Quantification of HKII normalized to γ-sarcoglycan and B6 CD quantities (N = 12, B6 CD verses MRL CD p = 0.000, B6 HFD versus MRL HFD p = 0.001, and B6 CD versus B6 HFD p = 0.013)

    Article Snippet: The slides where incubated with anti-γ sarcoglycan (1:100, Novocastra Labs, Buffalo Grove, IL, USA), anti-Glut4 (1:50, EMD Millipore, Darmstadt, Germany) or anti-fibronectin (1:100, Sigma, St. Louis, MO, USA) in PBS with 5 % fetal bovine serum for 1 h at room temperature, then aspirated and washed three times for 15 min in 4 °C PBS.

    Techniques:

    Mitochondrial differences in the two mouse strains. a The hearts from MRL mice contain increased mitochondrial genomes when normalized to nuclear DNA (N = 14, CD comparisons p = 0.002 and HFD comparisons p = 0.043). b However, quantification of VDAC immunoblots demonstrated no change in the outer mitochondrial membrane protein (N = 6). c Control to γ-sarcoglycan immunoblot. d VDAC normalized to γ-sarcoglycan (N = 6)

    Journal: Cardiovascular Diabetology

    Article Title: Successful metabolic adaptations leading to the prevention of high fat diet-induced murine cardiac remodeling

    doi: 10.1186/s12933-015-0286-0

    Figure Lengend Snippet: Mitochondrial differences in the two mouse strains. a The hearts from MRL mice contain increased mitochondrial genomes when normalized to nuclear DNA (N = 14, CD comparisons p = 0.002 and HFD comparisons p = 0.043). b However, quantification of VDAC immunoblots demonstrated no change in the outer mitochondrial membrane protein (N = 6). c Control to γ-sarcoglycan immunoblot. d VDAC normalized to γ-sarcoglycan (N = 6)

    Article Snippet: The slides where incubated with anti-γ sarcoglycan (1:100, Novocastra Labs, Buffalo Grove, IL, USA), anti-Glut4 (1:50, EMD Millipore, Darmstadt, Germany) or anti-fibronectin (1:100, Sigma, St. Louis, MO, USA) in PBS with 5 % fetal bovine serum for 1 h at room temperature, then aspirated and washed three times for 15 min in 4 °C PBS.

    Techniques: Mouse Assay, Western Blot

    Decreased MRL cardiac ACC indicates increased fatty acid metabolism. a pACC immunoblot. b Quantification normalized to γ-sarcoglycan shows the MRL tissues have reduced pACC (N = 6, B6 HFD versus MRL HFD p = 0.011). c ACC immunoblot. d Quantification normalized to γ-sarcoglycan shows the MRL tissues have reduced ACC (N = 6, B6 HFD versus MRL HFD p = 0.033, B6 CD versus B6 HFD p = 0.013)

    Journal: Cardiovascular Diabetology

    Article Title: Successful metabolic adaptations leading to the prevention of high fat diet-induced murine cardiac remodeling

    doi: 10.1186/s12933-015-0286-0

    Figure Lengend Snippet: Decreased MRL cardiac ACC indicates increased fatty acid metabolism. a pACC immunoblot. b Quantification normalized to γ-sarcoglycan shows the MRL tissues have reduced pACC (N = 6, B6 HFD versus MRL HFD p = 0.011). c ACC immunoblot. d Quantification normalized to γ-sarcoglycan shows the MRL tissues have reduced ACC (N = 6, B6 HFD versus MRL HFD p = 0.033, B6 CD versus B6 HFD p = 0.013)

    Article Snippet: The slides where incubated with anti-γ sarcoglycan (1:100, Novocastra Labs, Buffalo Grove, IL, USA), anti-Glut4 (1:50, EMD Millipore, Darmstadt, Germany) or anti-fibronectin (1:100, Sigma, St. Louis, MO, USA) in PBS with 5 % fetal bovine serum for 1 h at room temperature, then aspirated and washed three times for 15 min in 4 °C PBS.

    Techniques:

    The hearts from HFD B6 mice display morphologic pathologies. a The echocardiography determined left ventricular (LV) mass normalized to tibia length is larger in the HFD B6 mice when compared to the CD B6 mice ( p = 0.015, N > 8). b Similarly, the immunofluorescent determined cell size is larger in the HFD B6 mice when compared to the three other mouse groups (B6 CD versus B6 HFD, p = 0.025, B6 HFD versus MRL HFD p = 0.032, N > 3). c Representative cell sizes visualized by γ-sarcoglycan staining. d , e Echocardiography also revealed increases in left ventricle anterior wall during diastole and systole in the HFD B6 hearts compared to the CD B6 hearts (N > 8, bars indicate p

    Journal: Cardiovascular Diabetology

    Article Title: Successful metabolic adaptations leading to the prevention of high fat diet-induced murine cardiac remodeling

    doi: 10.1186/s12933-015-0286-0

    Figure Lengend Snippet: The hearts from HFD B6 mice display morphologic pathologies. a The echocardiography determined left ventricular (LV) mass normalized to tibia length is larger in the HFD B6 mice when compared to the CD B6 mice ( p = 0.015, N > 8). b Similarly, the immunofluorescent determined cell size is larger in the HFD B6 mice when compared to the three other mouse groups (B6 CD versus B6 HFD, p = 0.025, B6 HFD versus MRL HFD p = 0.032, N > 3). c Representative cell sizes visualized by γ-sarcoglycan staining. d , e Echocardiography also revealed increases in left ventricle anterior wall during diastole and systole in the HFD B6 hearts compared to the CD B6 hearts (N > 8, bars indicate p

    Article Snippet: The slides where incubated with anti-γ sarcoglycan (1:100, Novocastra Labs, Buffalo Grove, IL, USA), anti-Glut4 (1:50, EMD Millipore, Darmstadt, Germany) or anti-fibronectin (1:100, Sigma, St. Louis, MO, USA) in PBS with 5 % fetal bovine serum for 1 h at room temperature, then aspirated and washed three times for 15 min in 4 °C PBS.

    Techniques: Mouse Assay, Staining

    Glut4 is significantly reduced in the MRL hearts. a Representative immunoblot of Glut4 in mouse hearts. b γ-Sarcoglycan loading control. c Glut4 quantification normalized to γ-sarcoglycan (N = 9, B6 CD verses MRL CD p = 0.003, B6 HFD versus MRL HFD p = 0.001). d Representative immunofluorescence of Glut4 in the cardiac septum, original magnification ×100. e Brightness intensity of lines drawn ( white dashes in Fig. 5 d) across the plasma membranes

    Journal: Cardiovascular Diabetology

    Article Title: Successful metabolic adaptations leading to the prevention of high fat diet-induced murine cardiac remodeling

    doi: 10.1186/s12933-015-0286-0

    Figure Lengend Snippet: Glut4 is significantly reduced in the MRL hearts. a Representative immunoblot of Glut4 in mouse hearts. b γ-Sarcoglycan loading control. c Glut4 quantification normalized to γ-sarcoglycan (N = 9, B6 CD verses MRL CD p = 0.003, B6 HFD versus MRL HFD p = 0.001). d Representative immunofluorescence of Glut4 in the cardiac septum, original magnification ×100. e Brightness intensity of lines drawn ( white dashes in Fig. 5 d) across the plasma membranes

    Article Snippet: The slides where incubated with anti-γ sarcoglycan (1:100, Novocastra Labs, Buffalo Grove, IL, USA), anti-Glut4 (1:50, EMD Millipore, Darmstadt, Germany) or anti-fibronectin (1:100, Sigma, St. Louis, MO, USA) in PBS with 5 % fetal bovine serum for 1 h at room temperature, then aspirated and washed three times for 15 min in 4 °C PBS.

    Techniques: Immunofluorescence

    MRL hearts contain reduced amounts of pAMPK and AMPK. a Representative immunoblot of the phosphorylated α subunits of AMPK in mouse hearts. b γ-Sarcoglycan loading control. c pAMPK quantification normalized to γ-sarcoglycan and the B6 CD average (N = 15, B6 CD versus MRL CD p = 0.010, B6 HFD versus MRL HFD p = 0.025). d Representative immunoblot of the α subunits of AMPK in mouse hearts. e AMPK quantification normalized to the same γ-sarcoglycan blot as above and the CD B6 average (N = 9, B6 CD versus MRL CD p = 0.005, B6 HFD versus MRL HFD p = 0.038, MRL CD versus MRL HFD p = 0.037). f pAMPK/AMPK (N = 6)

    Journal: Cardiovascular Diabetology

    Article Title: Successful metabolic adaptations leading to the prevention of high fat diet-induced murine cardiac remodeling

    doi: 10.1186/s12933-015-0286-0

    Figure Lengend Snippet: MRL hearts contain reduced amounts of pAMPK and AMPK. a Representative immunoblot of the phosphorylated α subunits of AMPK in mouse hearts. b γ-Sarcoglycan loading control. c pAMPK quantification normalized to γ-sarcoglycan and the B6 CD average (N = 15, B6 CD versus MRL CD p = 0.010, B6 HFD versus MRL HFD p = 0.025). d Representative immunoblot of the α subunits of AMPK in mouse hearts. e AMPK quantification normalized to the same γ-sarcoglycan blot as above and the CD B6 average (N = 9, B6 CD versus MRL CD p = 0.005, B6 HFD versus MRL HFD p = 0.038, MRL CD versus MRL HFD p = 0.037). f pAMPK/AMPK (N = 6)

    Article Snippet: The slides where incubated with anti-γ sarcoglycan (1:100, Novocastra Labs, Buffalo Grove, IL, USA), anti-Glut4 (1:50, EMD Millipore, Darmstadt, Germany) or anti-fibronectin (1:100, Sigma, St. Louis, MO, USA) in PBS with 5 % fetal bovine serum for 1 h at room temperature, then aspirated and washed three times for 15 min in 4 °C PBS.

    Techniques:

    In vivo cross-linking of the sarcoglycans. Mouse myotubes were chemically cross-linked with 1 mM DTSSP and immunoprecipitated by anti–β-sarcoglycan antibody ( A ) or anti–α- or β-dystroglycan antibody ( B ). Lane 1 , cell lysate from mouse myotubes (26–34% input). Lanes 2 and 4 , immunoprecipitated products from uncross-linked myotubes. Lanes 3 and 5 , immunoprecipitated products from equal amount of DTSSP cross-linked myotubes. The composition of the immune complex was determined by Western blots using antibodies ( 1 ° Ab ) against different sarcoglycans and dystroglycans.

    Journal: The Journal of Cell Biology

    Article Title: Molecular Organization of Sarcoglycan Complex in Mouse Myotubes in Culture

    doi:

    Figure Lengend Snippet: In vivo cross-linking of the sarcoglycans. Mouse myotubes were chemically cross-linked with 1 mM DTSSP and immunoprecipitated by anti–β-sarcoglycan antibody ( A ) or anti–α- or β-dystroglycan antibody ( B ). Lane 1 , cell lysate from mouse myotubes (26–34% input). Lanes 2 and 4 , immunoprecipitated products from uncross-linked myotubes. Lanes 3 and 5 , immunoprecipitated products from equal amount of DTSSP cross-linked myotubes. The composition of the immune complex was determined by Western blots using antibodies ( 1 ° Ab ) against different sarcoglycans and dystroglycans.

    Article Snippet: Antibodies Mouse monoclonal antibodies directed against α-sarcoglycan (NCL–a-sarc); β-sarcoglycan (NCL–b-sarc), γ-sarcoglycan (NCL–g-sarc), δ-sarcoglycan (NCL–d-sarc), β-dystroglycan (NCL–b-DG), and dystrophin (NCL-dys2) were all purchased from Novocastra (Newcastle-upon-Tyne, UK) and each was diluted 1:100 for Western blotting and 1:200 for immunohistochemistry.

    Techniques: In Vivo, Immunoprecipitation, Western Blot

    Detection of intramolecular disulfide bond in the sarcoglycans. ( A ) Alignment of the carboxyl termini of β-, γ-, δ-, and C . elegans sarcoglycan with the consensus EGF-like repeat by MacVector Program (Oxford Molecular Group, Oxford, UK). Right , numbers correspond to the last amino acid residue in the protein; asterisk , stop codon; underline , four conserved cysteine residues. The cysteine 283 in γ-sarcoglycan ( italics ) is changed to tyrosine in patients with a severe form of early onset autosomal recessive muscular dystrophy (Piccolo et al., 1996). According to the consensus EGF-like repeat motif, the fifth cysteine is linked to the sixth cysteine by a disulfide bond ( Abe et al., 1998 ). This corresponds to the two middle conserved cysteine residues in the sarcoglycans. ( B ) Cell lysates from cultured mouse myotubes were electrophoresed in SDS-PAGE gel under nonreducing conditions (lane 1 ) and reducing conditions (lane 2 ). After transferred to nitrocellulose membranes, blots were examined by Western using antibodies ( 1 ° Ab ) against different sarcoglycans. The size of the sarcoglycans is indicated by a single arrow (nonreduced form) or a double arrow (reduced form). Note that the extra bands appearing on the nonreducing lane on the Western blot using the anti–β-sarcoglycan antibody were determined as nonspecific products unrelated to the DGC by 2-D diagonal gel electrophoresis (data not shown).

    Journal: The Journal of Cell Biology

    Article Title: Molecular Organization of Sarcoglycan Complex in Mouse Myotubes in Culture

    doi:

    Figure Lengend Snippet: Detection of intramolecular disulfide bond in the sarcoglycans. ( A ) Alignment of the carboxyl termini of β-, γ-, δ-, and C . elegans sarcoglycan with the consensus EGF-like repeat by MacVector Program (Oxford Molecular Group, Oxford, UK). Right , numbers correspond to the last amino acid residue in the protein; asterisk , stop codon; underline , four conserved cysteine residues. The cysteine 283 in γ-sarcoglycan ( italics ) is changed to tyrosine in patients with a severe form of early onset autosomal recessive muscular dystrophy (Piccolo et al., 1996). According to the consensus EGF-like repeat motif, the fifth cysteine is linked to the sixth cysteine by a disulfide bond ( Abe et al., 1998 ). This corresponds to the two middle conserved cysteine residues in the sarcoglycans. ( B ) Cell lysates from cultured mouse myotubes were electrophoresed in SDS-PAGE gel under nonreducing conditions (lane 1 ) and reducing conditions (lane 2 ). After transferred to nitrocellulose membranes, blots were examined by Western using antibodies ( 1 ° Ab ) against different sarcoglycans. The size of the sarcoglycans is indicated by a single arrow (nonreduced form) or a double arrow (reduced form). Note that the extra bands appearing on the nonreducing lane on the Western blot using the anti–β-sarcoglycan antibody were determined as nonspecific products unrelated to the DGC by 2-D diagonal gel electrophoresis (data not shown).

    Article Snippet: Antibodies Mouse monoclonal antibodies directed against α-sarcoglycan (NCL–a-sarc); β-sarcoglycan (NCL–b-sarc), γ-sarcoglycan (NCL–g-sarc), δ-sarcoglycan (NCL–d-sarc), β-dystroglycan (NCL–b-DG), and dystrophin (NCL-dys2) were all purchased from Novocastra (Newcastle-upon-Tyne, UK) and each was diluted 1:100 for Western blotting and 1:200 for immunohistochemistry.

    Techniques: Cell Culture, SDS Page, Western Blot, Nucleic Acid Electrophoresis

    Immunofluorescence of muscle biopsies from patients with autosomal recessive muscular dystrophy. Muscle section was stained with antibodies against dystrophin ( dys ), α-sarcoglycan ( α-sar ), β-sarcoglycan ( β-sar ), γ-sarcoglycan ( γ-sar ), and δ-sarcoglycan ( δ-sar ). Note that patients AL and CR showed different patterns of immunostaining for each of the sarcoglycans.

    Journal: The Journal of Cell Biology

    Article Title: Molecular Organization of Sarcoglycan Complex in Mouse Myotubes in Culture

    doi:

    Figure Lengend Snippet: Immunofluorescence of muscle biopsies from patients with autosomal recessive muscular dystrophy. Muscle section was stained with antibodies against dystrophin ( dys ), α-sarcoglycan ( α-sar ), β-sarcoglycan ( β-sar ), γ-sarcoglycan ( γ-sar ), and δ-sarcoglycan ( δ-sar ). Note that patients AL and CR showed different patterns of immunostaining for each of the sarcoglycans.

    Article Snippet: Antibodies Mouse monoclonal antibodies directed against α-sarcoglycan (NCL–a-sarc); β-sarcoglycan (NCL–b-sarc), γ-sarcoglycan (NCL–g-sarc), δ-sarcoglycan (NCL–d-sarc), β-dystroglycan (NCL–b-DG), and dystrophin (NCL-dys2) were all purchased from Novocastra (Newcastle-upon-Tyne, UK) and each was diluted 1:100 for Western blotting and 1:200 for immunohistochemistry.

    Techniques: Immunofluorescence, Staining, Immunostaining

    Coimmunoprecip-itation of the sarcoglycans under different stringencies. ( A ) Cell lysate from cultured mouse myotubes was immunoprecipitated by the anti– β-sarcoglycan antibody (NCL– b-sarc). The immune complex was washed in 1% NP-40 buffer containing no SDS (lane 2 ), 0.2% SDS (lane 3 ), 0.3% SDS (lane 4 ), and 0.4% SDS (lane 5 ). Lane 1 , cell lysate from mouse myotubes (33% input). (B) Immunoprecipitation was carried out after cultured mouse myotubes were lysed in 1% NP-40 buffer containing no SDS (lane 2 ), 0.1% SDS (lane 3 ), 0.2% SDS (lane 4 ), 0.3% SDS (lane 5 ), and 0.4% SDS (lane 6 ). Lane 1 , cell lysate from mouse myotubes (16% input). The final composition of the immune complex was determined by Western blots using antibodies ( 1 ° Ab ) against different sarcoglycans. Note that α-sarcoglycan was the most sensitive to increasing SDS concentration, resulting in dissociation from the complex in both cases.

    Journal: The Journal of Cell Biology

    Article Title: Molecular Organization of Sarcoglycan Complex in Mouse Myotubes in Culture

    doi:

    Figure Lengend Snippet: Coimmunoprecip-itation of the sarcoglycans under different stringencies. ( A ) Cell lysate from cultured mouse myotubes was immunoprecipitated by the anti– β-sarcoglycan antibody (NCL– b-sarc). The immune complex was washed in 1% NP-40 buffer containing no SDS (lane 2 ), 0.2% SDS (lane 3 ), 0.3% SDS (lane 4 ), and 0.4% SDS (lane 5 ). Lane 1 , cell lysate from mouse myotubes (33% input). (B) Immunoprecipitation was carried out after cultured mouse myotubes were lysed in 1% NP-40 buffer containing no SDS (lane 2 ), 0.1% SDS (lane 3 ), 0.2% SDS (lane 4 ), 0.3% SDS (lane 5 ), and 0.4% SDS (lane 6 ). Lane 1 , cell lysate from mouse myotubes (16% input). The final composition of the immune complex was determined by Western blots using antibodies ( 1 ° Ab ) against different sarcoglycans. Note that α-sarcoglycan was the most sensitive to increasing SDS concentration, resulting in dissociation from the complex in both cases.

    Article Snippet: Antibodies Mouse monoclonal antibodies directed against α-sarcoglycan (NCL–a-sarc); β-sarcoglycan (NCL–b-sarc), γ-sarcoglycan (NCL–g-sarc), δ-sarcoglycan (NCL–d-sarc), β-dystroglycan (NCL–b-DG), and dystrophin (NCL-dys2) were all purchased from Novocastra (Newcastle-upon-Tyne, UK) and each was diluted 1:100 for Western blotting and 1:200 for immunohistochemistry.

    Techniques: Cell Culture, Immunoprecipitation, Western Blot, Concentration Assay

    Analysis of the cross-linked sarcoglycans. Cell lysates from DTSSP cross-linked myotubes were examined by 2-D diagonal gel using antibodies against different sarcoglycans. ( A ) Schematic diagram of the principle of 2-D diagonal gel. Single circle , uncross-linked proteins; two circles joined by a line , cross-linked product. Shown in the examples are Western blots using antibodies against α-sarcoglycan, β- and α-dystroglycan ( B ), against β- and γ-sarcoglycan ( C ), and against β- and δ-sarcoglycan ( D ). Cross-linked proteins that appear below the diagonal line are represented by Arabic numerals: 1 , α-dystroglycan; 2 , β-dystroglycan; 4a and 4b , β-sarcoglycan; 5a , γ-sarcoglycan; and 6a , 6b , and 7 , δ-sarcoglycan. The estimated molecular weight of the three cross-linked products X, X 1 , and X 2 identified in this experiment are ∼200, 120, and 80 kD, respectively. Note that not all sarcoglycans and dystroglycans were cross-linked in the experiments. Uncross-linked proteins were found on the diagonal line and represented in Roman numerals: I , α-dystroglycan; II , β-dystroglycan; III , α-sarcoglycan; IV , β-sarcoglycan; V , γ-sarcoglycan; and VI , δ-sarcoglycan.

    Journal: The Journal of Cell Biology

    Article Title: Molecular Organization of Sarcoglycan Complex in Mouse Myotubes in Culture

    doi:

    Figure Lengend Snippet: Analysis of the cross-linked sarcoglycans. Cell lysates from DTSSP cross-linked myotubes were examined by 2-D diagonal gel using antibodies against different sarcoglycans. ( A ) Schematic diagram of the principle of 2-D diagonal gel. Single circle , uncross-linked proteins; two circles joined by a line , cross-linked product. Shown in the examples are Western blots using antibodies against α-sarcoglycan, β- and α-dystroglycan ( B ), against β- and γ-sarcoglycan ( C ), and against β- and δ-sarcoglycan ( D ). Cross-linked proteins that appear below the diagonal line are represented by Arabic numerals: 1 , α-dystroglycan; 2 , β-dystroglycan; 4a and 4b , β-sarcoglycan; 5a , γ-sarcoglycan; and 6a , 6b , and 7 , δ-sarcoglycan. The estimated molecular weight of the three cross-linked products X, X 1 , and X 2 identified in this experiment are ∼200, 120, and 80 kD, respectively. Note that not all sarcoglycans and dystroglycans were cross-linked in the experiments. Uncross-linked proteins were found on the diagonal line and represented in Roman numerals: I , α-dystroglycan; II , β-dystroglycan; III , α-sarcoglycan; IV , β-sarcoglycan; V , γ-sarcoglycan; and VI , δ-sarcoglycan.

    Article Snippet: Antibodies Mouse monoclonal antibodies directed against α-sarcoglycan (NCL–a-sarc); β-sarcoglycan (NCL–b-sarc), γ-sarcoglycan (NCL–g-sarc), δ-sarcoglycan (NCL–d-sarc), β-dystroglycan (NCL–b-DG), and dystrophin (NCL-dys2) were all purchased from Novocastra (Newcastle-upon-Tyne, UK) and each was diluted 1:100 for Western blotting and 1:200 for immunohistochemistry.

    Techniques: Western Blot, Molecular Weight

    Structural model of the sarcoglycan complex and the dystroglycan complex. The four sarcoglycans ( left ) are represented by α, β, γ, and δ. α-DG and β-DG denote α- and β-dystroglycan ( right ), respectively. Branch structure corresponds to N-glycoside sugar chain. SH , disulfide linkage. Double-headed arrow , potential interaction betw een δ-sarcoglycan and the dystroglycan complex. In the model, β-sarcoglycan is tightly associated with δ-sarcoglycan. α-Sarcoglycan is placed apart from other sarcoglycans and is viewed as a separate subunit within the sarcoglycan complex.

    Journal: The Journal of Cell Biology

    Article Title: Molecular Organization of Sarcoglycan Complex in Mouse Myotubes in Culture

    doi:

    Figure Lengend Snippet: Structural model of the sarcoglycan complex and the dystroglycan complex. The four sarcoglycans ( left ) are represented by α, β, γ, and δ. α-DG and β-DG denote α- and β-dystroglycan ( right ), respectively. Branch structure corresponds to N-glycoside sugar chain. SH , disulfide linkage. Double-headed arrow , potential interaction betw een δ-sarcoglycan and the dystroglycan complex. In the model, β-sarcoglycan is tightly associated with δ-sarcoglycan. α-Sarcoglycan is placed apart from other sarcoglycans and is viewed as a separate subunit within the sarcoglycan complex.

    Article Snippet: Antibodies Mouse monoclonal antibodies directed against α-sarcoglycan (NCL–a-sarc); β-sarcoglycan (NCL–b-sarc), γ-sarcoglycan (NCL–g-sarc), δ-sarcoglycan (NCL–d-sarc), β-dystroglycan (NCL–b-DG), and dystrophin (NCL-dys2) were all purchased from Novocastra (Newcastle-upon-Tyne, UK) and each was diluted 1:100 for Western blotting and 1:200 for immunohistochemistry.

    Techniques:

    Hexokinase II is also reduced in the MRL cardiac tissue. a Representative immunoblot of HKII in the heart tissues. b γ-Sarcoglycan loading control. c Quantification of HKII normalized to γ-sarcoglycan and B6 CD quantities (N = 12, B6 CD verses MRL CD p = 0.000, B6 HFD versus MRL HFD p = 0.001, and B6 CD versus B6 HFD p = 0.013)

    Journal: Cardiovascular Diabetology

    Article Title: Successful metabolic adaptations leading to the prevention of high fat diet-induced murine cardiac remodeling

    doi: 10.1186/s12933-015-0286-0

    Figure Lengend Snippet: Hexokinase II is also reduced in the MRL cardiac tissue. a Representative immunoblot of HKII in the heart tissues. b γ-Sarcoglycan loading control. c Quantification of HKII normalized to γ-sarcoglycan and B6 CD quantities (N = 12, B6 CD verses MRL CD p = 0.000, B6 HFD versus MRL HFD p = 0.001, and B6 CD versus B6 HFD p = 0.013)

    Article Snippet: The membranes were incubated at 4 °C overnight in 5 % NFDM/TBST with primary antibodies [anti-AMPK, anti-pAMPK, and PGC-1α (1:1000, Cell Signaling Technology, Danvers, MA, USA); anti-ACC, anti-pACC, fibronectin (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-HKII (1:5000, Millipore, Billerica, MA, USA); VDAC, Oxidative Phosphorylation antibody cocktail (1:1000, Abcam, Cambridge, MA, USA), Glut4 (1:500, EMD Millipore, Darmstadt, Germany), and γ-sarcoglycan (1:1000, Novocastra Labs, Buffalo Grove, IL, USA)].

    Techniques:

    Mitochondrial differences in the two mouse strains. a The hearts from MRL mice contain increased mitochondrial genomes when normalized to nuclear DNA (N = 14, CD comparisons p = 0.002 and HFD comparisons p = 0.043). b However, quantification of VDAC immunoblots demonstrated no change in the outer mitochondrial membrane protein (N = 6). c Control to γ-sarcoglycan immunoblot. d VDAC normalized to γ-sarcoglycan (N = 6)

    Journal: Cardiovascular Diabetology

    Article Title: Successful metabolic adaptations leading to the prevention of high fat diet-induced murine cardiac remodeling

    doi: 10.1186/s12933-015-0286-0

    Figure Lengend Snippet: Mitochondrial differences in the two mouse strains. a The hearts from MRL mice contain increased mitochondrial genomes when normalized to nuclear DNA (N = 14, CD comparisons p = 0.002 and HFD comparisons p = 0.043). b However, quantification of VDAC immunoblots demonstrated no change in the outer mitochondrial membrane protein (N = 6). c Control to γ-sarcoglycan immunoblot. d VDAC normalized to γ-sarcoglycan (N = 6)

    Article Snippet: The membranes were incubated at 4 °C overnight in 5 % NFDM/TBST with primary antibodies [anti-AMPK, anti-pAMPK, and PGC-1α (1:1000, Cell Signaling Technology, Danvers, MA, USA); anti-ACC, anti-pACC, fibronectin (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-HKII (1:5000, Millipore, Billerica, MA, USA); VDAC, Oxidative Phosphorylation antibody cocktail (1:1000, Abcam, Cambridge, MA, USA), Glut4 (1:500, EMD Millipore, Darmstadt, Germany), and γ-sarcoglycan (1:1000, Novocastra Labs, Buffalo Grove, IL, USA)].

    Techniques: Mouse Assay, Western Blot

    Decreased MRL cardiac ACC indicates increased fatty acid metabolism. a pACC immunoblot. b Quantification normalized to γ-sarcoglycan shows the MRL tissues have reduced pACC (N = 6, B6 HFD versus MRL HFD p = 0.011). c ACC immunoblot. d Quantification normalized to γ-sarcoglycan shows the MRL tissues have reduced ACC (N = 6, B6 HFD versus MRL HFD p = 0.033, B6 CD versus B6 HFD p = 0.013)

    Journal: Cardiovascular Diabetology

    Article Title: Successful metabolic adaptations leading to the prevention of high fat diet-induced murine cardiac remodeling

    doi: 10.1186/s12933-015-0286-0

    Figure Lengend Snippet: Decreased MRL cardiac ACC indicates increased fatty acid metabolism. a pACC immunoblot. b Quantification normalized to γ-sarcoglycan shows the MRL tissues have reduced pACC (N = 6, B6 HFD versus MRL HFD p = 0.011). c ACC immunoblot. d Quantification normalized to γ-sarcoglycan shows the MRL tissues have reduced ACC (N = 6, B6 HFD versus MRL HFD p = 0.033, B6 CD versus B6 HFD p = 0.013)

    Article Snippet: The membranes were incubated at 4 °C overnight in 5 % NFDM/TBST with primary antibodies [anti-AMPK, anti-pAMPK, and PGC-1α (1:1000, Cell Signaling Technology, Danvers, MA, USA); anti-ACC, anti-pACC, fibronectin (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-HKII (1:5000, Millipore, Billerica, MA, USA); VDAC, Oxidative Phosphorylation antibody cocktail (1:1000, Abcam, Cambridge, MA, USA), Glut4 (1:500, EMD Millipore, Darmstadt, Germany), and γ-sarcoglycan (1:1000, Novocastra Labs, Buffalo Grove, IL, USA)].

    Techniques:

    The hearts from HFD B6 mice display morphologic pathologies. a The echocardiography determined left ventricular (LV) mass normalized to tibia length is larger in the HFD B6 mice when compared to the CD B6 mice ( p = 0.015, N > 8). b Similarly, the immunofluorescent determined cell size is larger in the HFD B6 mice when compared to the three other mouse groups (B6 CD versus B6 HFD, p = 0.025, B6 HFD versus MRL HFD p = 0.032, N > 3). c Representative cell sizes visualized by γ-sarcoglycan staining. d , e Echocardiography also revealed increases in left ventricle anterior wall during diastole and systole in the HFD B6 hearts compared to the CD B6 hearts (N > 8, bars indicate p

    Journal: Cardiovascular Diabetology

    Article Title: Successful metabolic adaptations leading to the prevention of high fat diet-induced murine cardiac remodeling

    doi: 10.1186/s12933-015-0286-0

    Figure Lengend Snippet: The hearts from HFD B6 mice display morphologic pathologies. a The echocardiography determined left ventricular (LV) mass normalized to tibia length is larger in the HFD B6 mice when compared to the CD B6 mice ( p = 0.015, N > 8). b Similarly, the immunofluorescent determined cell size is larger in the HFD B6 mice when compared to the three other mouse groups (B6 CD versus B6 HFD, p = 0.025, B6 HFD versus MRL HFD p = 0.032, N > 3). c Representative cell sizes visualized by γ-sarcoglycan staining. d , e Echocardiography also revealed increases in left ventricle anterior wall during diastole and systole in the HFD B6 hearts compared to the CD B6 hearts (N > 8, bars indicate p

    Article Snippet: The membranes were incubated at 4 °C overnight in 5 % NFDM/TBST with primary antibodies [anti-AMPK, anti-pAMPK, and PGC-1α (1:1000, Cell Signaling Technology, Danvers, MA, USA); anti-ACC, anti-pACC, fibronectin (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-HKII (1:5000, Millipore, Billerica, MA, USA); VDAC, Oxidative Phosphorylation antibody cocktail (1:1000, Abcam, Cambridge, MA, USA), Glut4 (1:500, EMD Millipore, Darmstadt, Germany), and γ-sarcoglycan (1:1000, Novocastra Labs, Buffalo Grove, IL, USA)].

    Techniques: Mouse Assay, Staining

    Glut4 is significantly reduced in the MRL hearts. a Representative immunoblot of Glut4 in mouse hearts. b γ-Sarcoglycan loading control. c Glut4 quantification normalized to γ-sarcoglycan (N = 9, B6 CD verses MRL CD p = 0.003, B6 HFD versus MRL HFD p = 0.001). d Representative immunofluorescence of Glut4 in the cardiac septum, original magnification ×100. e Brightness intensity of lines drawn ( white dashes in Fig. 5 d) across the plasma membranes

    Journal: Cardiovascular Diabetology

    Article Title: Successful metabolic adaptations leading to the prevention of high fat diet-induced murine cardiac remodeling

    doi: 10.1186/s12933-015-0286-0

    Figure Lengend Snippet: Glut4 is significantly reduced in the MRL hearts. a Representative immunoblot of Glut4 in mouse hearts. b γ-Sarcoglycan loading control. c Glut4 quantification normalized to γ-sarcoglycan (N = 9, B6 CD verses MRL CD p = 0.003, B6 HFD versus MRL HFD p = 0.001). d Representative immunofluorescence of Glut4 in the cardiac septum, original magnification ×100. e Brightness intensity of lines drawn ( white dashes in Fig. 5 d) across the plasma membranes

    Article Snippet: The membranes were incubated at 4 °C overnight in 5 % NFDM/TBST with primary antibodies [anti-AMPK, anti-pAMPK, and PGC-1α (1:1000, Cell Signaling Technology, Danvers, MA, USA); anti-ACC, anti-pACC, fibronectin (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-HKII (1:5000, Millipore, Billerica, MA, USA); VDAC, Oxidative Phosphorylation antibody cocktail (1:1000, Abcam, Cambridge, MA, USA), Glut4 (1:500, EMD Millipore, Darmstadt, Germany), and γ-sarcoglycan (1:1000, Novocastra Labs, Buffalo Grove, IL, USA)].

    Techniques: Immunofluorescence

    MRL hearts contain reduced amounts of pAMPK and AMPK. a Representative immunoblot of the phosphorylated α subunits of AMPK in mouse hearts. b γ-Sarcoglycan loading control. c pAMPK quantification normalized to γ-sarcoglycan and the B6 CD average (N = 15, B6 CD versus MRL CD p = 0.010, B6 HFD versus MRL HFD p = 0.025). d Representative immunoblot of the α subunits of AMPK in mouse hearts. e AMPK quantification normalized to the same γ-sarcoglycan blot as above and the CD B6 average (N = 9, B6 CD versus MRL CD p = 0.005, B6 HFD versus MRL HFD p = 0.038, MRL CD versus MRL HFD p = 0.037). f pAMPK/AMPK (N = 6)

    Journal: Cardiovascular Diabetology

    Article Title: Successful metabolic adaptations leading to the prevention of high fat diet-induced murine cardiac remodeling

    doi: 10.1186/s12933-015-0286-0

    Figure Lengend Snippet: MRL hearts contain reduced amounts of pAMPK and AMPK. a Representative immunoblot of the phosphorylated α subunits of AMPK in mouse hearts. b γ-Sarcoglycan loading control. c pAMPK quantification normalized to γ-sarcoglycan and the B6 CD average (N = 15, B6 CD versus MRL CD p = 0.010, B6 HFD versus MRL HFD p = 0.025). d Representative immunoblot of the α subunits of AMPK in mouse hearts. e AMPK quantification normalized to the same γ-sarcoglycan blot as above and the CD B6 average (N = 9, B6 CD versus MRL CD p = 0.005, B6 HFD versus MRL HFD p = 0.038, MRL CD versus MRL HFD p = 0.037). f pAMPK/AMPK (N = 6)

    Article Snippet: The membranes were incubated at 4 °C overnight in 5 % NFDM/TBST with primary antibodies [anti-AMPK, anti-pAMPK, and PGC-1α (1:1000, Cell Signaling Technology, Danvers, MA, USA); anti-ACC, anti-pACC, fibronectin (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-HKII (1:5000, Millipore, Billerica, MA, USA); VDAC, Oxidative Phosphorylation antibody cocktail (1:1000, Abcam, Cambridge, MA, USA), Glut4 (1:500, EMD Millipore, Darmstadt, Germany), and γ-sarcoglycan (1:1000, Novocastra Labs, Buffalo Grove, IL, USA)].

    Techniques:

    Absence of intermolecular disulfide bonds between the sarcoglycans. Cell lysate from cultured mouse myotubes was electrophoresed in 2-D diagonal gel and examined by Western blots using antibodies against different sarcoglycans. Shown in the example is the Western blot using antibodies against α-, β-, and δ-sarcoglycan. Note that no extra spot was observed below the diagonal line ( dashed line ).

    Journal: The Journal of Cell Biology

    Article Title: Molecular Organization of Sarcoglycan Complex in Mouse Myotubes in Culture

    doi:

    Figure Lengend Snippet: Absence of intermolecular disulfide bonds between the sarcoglycans. Cell lysate from cultured mouse myotubes was electrophoresed in 2-D diagonal gel and examined by Western blots using antibodies against different sarcoglycans. Shown in the example is the Western blot using antibodies against α-, β-, and δ-sarcoglycan. Note that no extra spot was observed below the diagonal line ( dashed line ).

    Article Snippet: Antibodies Mouse monoclonal antibodies directed against α-sarcoglycan (NCL–a-sarc); β-sarcoglycan (NCL–b-sarc), γ-sarcoglycan (NCL–g-sarc), δ-sarcoglycan (NCL–d-sarc), β-dystroglycan (NCL–b-DG), and dystrophin (NCL-dys2) were all purchased from Novocastra (Newcastle-upon-Tyne, UK) and each was diluted 1:100 for Western blotting and 1:200 for immunohistochemistry.

    Techniques: Cell Culture, Western Blot

    Immunofluorescence of muscle biopsies from patients with autosomal recessive muscular dystrophy. Muscle section was stained with antibodies against dystrophin ( dys ), α-sarcoglycan ( α-sar ), β-sarcoglycan ( β-sar ), γ-sarcoglycan ( γ-sar ), and δ-sarcoglycan ( δ-sar ). Note that patients AL and CR showed different patterns of immunostaining for each of the sarcoglycans.

    Journal: The Journal of Cell Biology

    Article Title: Molecular Organization of Sarcoglycan Complex in Mouse Myotubes in Culture

    doi:

    Figure Lengend Snippet: Immunofluorescence of muscle biopsies from patients with autosomal recessive muscular dystrophy. Muscle section was stained with antibodies against dystrophin ( dys ), α-sarcoglycan ( α-sar ), β-sarcoglycan ( β-sar ), γ-sarcoglycan ( γ-sar ), and δ-sarcoglycan ( δ-sar ). Note that patients AL and CR showed different patterns of immunostaining for each of the sarcoglycans.

    Article Snippet: Antibodies Mouse monoclonal antibodies directed against α-sarcoglycan (NCL–a-sarc); β-sarcoglycan (NCL–b-sarc), γ-sarcoglycan (NCL–g-sarc), δ-sarcoglycan (NCL–d-sarc), β-dystroglycan (NCL–b-DG), and dystrophin (NCL-dys2) were all purchased from Novocastra (Newcastle-upon-Tyne, UK) and each was diluted 1:100 for Western blotting and 1:200 for immunohistochemistry.

    Techniques: Immunofluorescence, Staining, Immunostaining

    Coimmunoprecipitation of the sarcoglycans using different anti-sarcoglycan antibodies. Cell lysate from cultured mouse myotubes were immunoprecipitated by an antibody directed against α-, β-, γ-, or δ-sarcoglycan. The immune complex was analyzed by Western blots using antibodies ( 1 ° Ab ) against different sarcoglycans. Lane 1 , cell lysate from mouse myotubes ( 13–16% input ). Lane 2 , immunoprecipitated products. Note that different sarcoglycans were precipitated by different anti-sarcoglycan antibodies.

    Journal: The Journal of Cell Biology

    Article Title: Molecular Organization of Sarcoglycan Complex in Mouse Myotubes in Culture

    doi:

    Figure Lengend Snippet: Coimmunoprecipitation of the sarcoglycans using different anti-sarcoglycan antibodies. Cell lysate from cultured mouse myotubes were immunoprecipitated by an antibody directed against α-, β-, γ-, or δ-sarcoglycan. The immune complex was analyzed by Western blots using antibodies ( 1 ° Ab ) against different sarcoglycans. Lane 1 , cell lysate from mouse myotubes ( 13–16% input ). Lane 2 , immunoprecipitated products. Note that different sarcoglycans were precipitated by different anti-sarcoglycan antibodies.

    Article Snippet: Antibodies Mouse monoclonal antibodies directed against α-sarcoglycan (NCL–a-sarc); β-sarcoglycan (NCL–b-sarc), γ-sarcoglycan (NCL–g-sarc), δ-sarcoglycan (NCL–d-sarc), β-dystroglycan (NCL–b-DG), and dystrophin (NCL-dys2) were all purchased from Novocastra (Newcastle-upon-Tyne, UK) and each was diluted 1:100 for Western blotting and 1:200 for immunohistochemistry.

    Techniques: Cell Culture, Immunoprecipitation, Western Blot

    Analysis of the cross-linked sarcoglycans. Cell lysates from DTSSP cross-linked myotubes were examined by 2-D diagonal gel using antibodies against different sarcoglycans. ( A ) Schematic diagram of the principle of 2-D diagonal gel. Single circle , uncross-linked proteins; two circles joined by a line , cross-linked product. Shown in the examples are Western blots using antibodies against α-sarcoglycan, β- and α-dystroglycan ( B ), against β- and γ-sarcoglycan ( C ), and against β- and δ-sarcoglycan ( D ). Cross-linked proteins that appear below the diagonal line are represented by Arabic numerals: 1 , α-dystroglycan; 2 , β-dystroglycan; 4a and 4b , β-sarcoglycan; 5a , γ-sarcoglycan; and 6a , 6b , and 7 , δ-sarcoglycan. The estimated molecular weight of the three cross-linked products X, X 1 , and X 2 identified in this experiment are ∼200, 120, and 80 kD, respectively. Note that not all sarcoglycans and dystroglycans were cross-linked in the experiments. Uncross-linked proteins were found on the diagonal line and represented in Roman numerals: I , α-dystroglycan; II , β-dystroglycan; III , α-sarcoglycan; IV , β-sarcoglycan; V , γ-sarcoglycan; and VI , δ-sarcoglycan.

    Journal: The Journal of Cell Biology

    Article Title: Molecular Organization of Sarcoglycan Complex in Mouse Myotubes in Culture

    doi:

    Figure Lengend Snippet: Analysis of the cross-linked sarcoglycans. Cell lysates from DTSSP cross-linked myotubes were examined by 2-D diagonal gel using antibodies against different sarcoglycans. ( A ) Schematic diagram of the principle of 2-D diagonal gel. Single circle , uncross-linked proteins; two circles joined by a line , cross-linked product. Shown in the examples are Western blots using antibodies against α-sarcoglycan, β- and α-dystroglycan ( B ), against β- and γ-sarcoglycan ( C ), and against β- and δ-sarcoglycan ( D ). Cross-linked proteins that appear below the diagonal line are represented by Arabic numerals: 1 , α-dystroglycan; 2 , β-dystroglycan; 4a and 4b , β-sarcoglycan; 5a , γ-sarcoglycan; and 6a , 6b , and 7 , δ-sarcoglycan. The estimated molecular weight of the three cross-linked products X, X 1 , and X 2 identified in this experiment are ∼200, 120, and 80 kD, respectively. Note that not all sarcoglycans and dystroglycans were cross-linked in the experiments. Uncross-linked proteins were found on the diagonal line and represented in Roman numerals: I , α-dystroglycan; II , β-dystroglycan; III , α-sarcoglycan; IV , β-sarcoglycan; V , γ-sarcoglycan; and VI , δ-sarcoglycan.

    Article Snippet: Antibodies Mouse monoclonal antibodies directed against α-sarcoglycan (NCL–a-sarc); β-sarcoglycan (NCL–b-sarc), γ-sarcoglycan (NCL–g-sarc), δ-sarcoglycan (NCL–d-sarc), β-dystroglycan (NCL–b-DG), and dystrophin (NCL-dys2) were all purchased from Novocastra (Newcastle-upon-Tyne, UK) and each was diluted 1:100 for Western blotting and 1:200 for immunohistochemistry.

    Techniques: Western Blot, Molecular Weight

    Structural model of the sarcoglycan complex and the dystroglycan complex. The four sarcoglycans ( left ) are represented by α, β, γ, and δ. α-DG and β-DG denote α- and β-dystroglycan ( right ), respectively. Branch structure corresponds to N-glycoside sugar chain. SH , disulfide linkage. Double-headed arrow , potential interaction betw een δ-sarcoglycan and the dystroglycan complex. In the model, β-sarcoglycan is tightly associated with δ-sarcoglycan. α-Sarcoglycan is placed apart from other sarcoglycans and is viewed as a separate subunit within the sarcoglycan complex.

    Journal: The Journal of Cell Biology

    Article Title: Molecular Organization of Sarcoglycan Complex in Mouse Myotubes in Culture

    doi:

    Figure Lengend Snippet: Structural model of the sarcoglycan complex and the dystroglycan complex. The four sarcoglycans ( left ) are represented by α, β, γ, and δ. α-DG and β-DG denote α- and β-dystroglycan ( right ), respectively. Branch structure corresponds to N-glycoside sugar chain. SH , disulfide linkage. Double-headed arrow , potential interaction betw een δ-sarcoglycan and the dystroglycan complex. In the model, β-sarcoglycan is tightly associated with δ-sarcoglycan. α-Sarcoglycan is placed apart from other sarcoglycans and is viewed as a separate subunit within the sarcoglycan complex.

    Article Snippet: Antibodies Mouse monoclonal antibodies directed against α-sarcoglycan (NCL–a-sarc); β-sarcoglycan (NCL–b-sarc), γ-sarcoglycan (NCL–g-sarc), δ-sarcoglycan (NCL–d-sarc), β-dystroglycan (NCL–b-DG), and dystrophin (NCL-dys2) were all purchased from Novocastra (Newcastle-upon-Tyne, UK) and each was diluted 1:100 for Western blotting and 1:200 for immunohistochemistry.

    Techniques:

    Disruption of dystrophin expression in the skeletal muscle of DMD KO rabbits. (A-C) Immunofluorescence staining of muscle sections from WT and DMD KO rabbits with mouse monoclonal antibodies against dystrophin (A), glycosylated α-dystroglycan (B) and α-sarcoglycan (C). Nuclei were stained by DAPI. Scale bar: 100 µm.

    Journal: Disease Models & Mechanisms

    Article Title: A novel rabbit model of Duchenne muscular dystrophy generated by CRISPR/Cas9

    doi: 10.1242/dmm.032201

    Figure Lengend Snippet: Disruption of dystrophin expression in the skeletal muscle of DMD KO rabbits. (A-C) Immunofluorescence staining of muscle sections from WT and DMD KO rabbits with mouse monoclonal antibodies against dystrophin (A), glycosylated α-dystroglycan (B) and α-sarcoglycan (C). Nuclei were stained by DAPI. Scale bar: 100 µm.

    Article Snippet: Primary antibodies against dystrophin (MANDYS1 clone 3B7, 1:20, Developmental Studies Hybridoma Bank), α-dystroglycan (sc-53987, 1:50, Santa Cruz Biotechnology), α-sarcoglycan (NCL-L-a-SARC, 1:100, Leica Biosystems) and caveolin 3 (610420, 1:500, BD Biosciences) were used.

    Techniques: Expressing, Immunofluorescence, Staining

    Tsp expression in muscle of Drosophila rescues MD due to deletion of the δ-sarcoglycan-like gene (Sgcd 840 ). ( A ) Fly survival was compared over a period of 40 days. p

    Journal: eLife

    Article Title: Thrombospondin expression in myofibers stabilizes muscle membranes

    doi: 10.7554/eLife.17589

    Figure Lengend Snippet: Tsp expression in muscle of Drosophila rescues MD due to deletion of the δ-sarcoglycan-like gene (Sgcd 840 ). ( A ) Fly survival was compared over a period of 40 days. p

    Article Snippet: Primary antibodies included: δ-sarcoglycan (Abcam, ab92896, 1:100), α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan (NovaCastra, Buffalo Grove, IL, NCL-a-sarc, NCL-b-sarc and NCL-g-sarc, all 1:250), β-dystroglycan (Development Studies Hybridoma Bank, Iowa City IA, MANDAG2; 1:50), utrophin (Santa Cruz Biotechnology, MANCHO7, sc-81557; 1:50), dystrophin (Abcam, ab15277, 1:200) and β1D-integrin (Millipore, Billerica, MA, MAB1900, 1:250).

    Techniques: Expressing

    Thbs4 enhances stabilizing proteins at the sarcolemma and directly interacts with integrins. ( A , B ) Representative Western blots of sarcolemmal protein extracts from the quadriceps of the indicated groups of mice at three months of age (n = 4–5 biological replicates). Sgcd -/- Tg and mdx Tg indicate Sgcd -/- and mdx with skeletal muscle specific Thbs4 overexpression, respectively. Ponceau staining of a nonspecific band and dihydropyridine receptor α1 (Cav1.1) were used as loading controls. Abbreviations: Utro, utrophin; Dystro, dystrophin; Dysfer, dysferlin; α-DG, α-dystroglycan; β-DG, β-dystroglycan; δ-SCG, δ-sarcoglycan; α-SCG, α-sarcoglycan; β-SGC, β-sarcoglycan; β1D-, α7- and α5-itg (integrin). ( C ) Immunoblots for β1D- and α7-Integrin (Itg), β-dystroglycan (DG) and Thbs4 (Flag) from neonatal rat ventricular myocyte extracts immunoprecipitated with a Flag antibody (Thbs4). Adβgal was used as a control infection (n = 3 biological replicates). An adenovirus expressing a Flag-tagged Thbs4 protein was used to achieve high level of this protein to identify the interaction. ( D , E ) Representative Western blots for Thbs4, α5- and β1D-integrin (itg) from intracellular vesicular isolates from WT and Thbs4 Tg quadriceps ( D ) or WT and Sgcd -/- quadriceps ( E ) that were immunoprecipitated with an antibody raised against the cytoplasmic domain of β1D-integrin (n = 3 biological replicates), showing that Thbs4 and α5-integrin localize to β1D-integrin-positive intracellular vesicles. α-tubulin and Gapdh are presented as loading control. DOI: http://dx.doi.org/10.7554/eLife.17589.019

    Journal: eLife

    Article Title: Thrombospondin expression in myofibers stabilizes muscle membranes

    doi: 10.7554/eLife.17589

    Figure Lengend Snippet: Thbs4 enhances stabilizing proteins at the sarcolemma and directly interacts with integrins. ( A , B ) Representative Western blots of sarcolemmal protein extracts from the quadriceps of the indicated groups of mice at three months of age (n = 4–5 biological replicates). Sgcd -/- Tg and mdx Tg indicate Sgcd -/- and mdx with skeletal muscle specific Thbs4 overexpression, respectively. Ponceau staining of a nonspecific band and dihydropyridine receptor α1 (Cav1.1) were used as loading controls. Abbreviations: Utro, utrophin; Dystro, dystrophin; Dysfer, dysferlin; α-DG, α-dystroglycan; β-DG, β-dystroglycan; δ-SCG, δ-sarcoglycan; α-SCG, α-sarcoglycan; β-SGC, β-sarcoglycan; β1D-, α7- and α5-itg (integrin). ( C ) Immunoblots for β1D- and α7-Integrin (Itg), β-dystroglycan (DG) and Thbs4 (Flag) from neonatal rat ventricular myocyte extracts immunoprecipitated with a Flag antibody (Thbs4). Adβgal was used as a control infection (n = 3 biological replicates). An adenovirus expressing a Flag-tagged Thbs4 protein was used to achieve high level of this protein to identify the interaction. ( D , E ) Representative Western blots for Thbs4, α5- and β1D-integrin (itg) from intracellular vesicular isolates from WT and Thbs4 Tg quadriceps ( D ) or WT and Sgcd -/- quadriceps ( E ) that were immunoprecipitated with an antibody raised against the cytoplasmic domain of β1D-integrin (n = 3 biological replicates), showing that Thbs4 and α5-integrin localize to β1D-integrin-positive intracellular vesicles. α-tubulin and Gapdh are presented as loading control. DOI: http://dx.doi.org/10.7554/eLife.17589.019

    Article Snippet: Primary antibodies included: δ-sarcoglycan (Abcam, ab92896, 1:100), α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan (NovaCastra, Buffalo Grove, IL, NCL-a-sarc, NCL-b-sarc and NCL-g-sarc, all 1:250), β-dystroglycan (Development Studies Hybridoma Bank, Iowa City IA, MANDAG2; 1:50), utrophin (Santa Cruz Biotechnology, MANCHO7, sc-81557; 1:50), dystrophin (Abcam, ab15277, 1:200) and β1D-integrin (Millipore, Billerica, MA, MAB1900, 1:250).

    Techniques: Western Blot, Mouse Assay, Over Expression, Staining, Immunoprecipitation, Infection, Expressing

    Generation of dystrophin/δ-sarcoglycan double mutant mice. ( A ) The breeding scheme. ( B ) Representative PCR genotyping results. Bkg, background; δSG KO, δ-sarcoglycan deficient mice; δ-Dko, δ-sarcoglycan/dystrophin

    Journal: Human Molecular Genetics

    Article Title: Sub-physiological sarcoglycan expression contributes to compensatory muscle protection in mdx mice

    doi: 10.1093/hmg/ddp015

    Figure Lengend Snippet: Generation of dystrophin/δ-sarcoglycan double mutant mice. ( A ) The breeding scheme. ( B ) Representative PCR genotyping results. Bkg, background; δSG KO, δ-sarcoglycan deficient mice; δ-Dko, δ-sarcoglycan/dystrophin

    Article Snippet: Utrophin was examined with a mouse monoclonal antibody against the utrophin N-terminal domain (VP-U579, 1:20; clone DRP3/20C5, IgG1; Vector Laboratories, Burlingame, CA). β-Dystroglycan was revealed with NCL-b-DG (1:50; Novocastra). α-Sarcoglycan was detected with VP-A105 (1:50; Vector Laboratories). β-Sarcoglycan was revealed with NCL-b-SARC (1:50; Novocastra). γ-Sarcoglycan was detected with VP-G803 (1:50; Vector Laboratories). δ-Sarcoglycan was detected with sc28281 (1:50; Santa Cruz Biotechnology Inc.).

    Techniques: Mutagenesis, Mouse Assay, Polymerase Chain Reaction

    Dystrophin/δ-sarcoglycan double knockout mice display a severer clinical phenotype. ( A ) Kyphosis (arrow) and hind limb joint contracture were apparent in δ-Dko mice. ( B ) Body weights of 3-month-old male BL10, mdx, δSG KO and δ-Dko

    Journal: Human Molecular Genetics

    Article Title: Sub-physiological sarcoglycan expression contributes to compensatory muscle protection in mdx mice

    doi: 10.1093/hmg/ddp015

    Figure Lengend Snippet: Dystrophin/δ-sarcoglycan double knockout mice display a severer clinical phenotype. ( A ) Kyphosis (arrow) and hind limb joint contracture were apparent in δ-Dko mice. ( B ) Body weights of 3-month-old male BL10, mdx, δSG KO and δ-Dko

    Article Snippet: Utrophin was examined with a mouse monoclonal antibody against the utrophin N-terminal domain (VP-U579, 1:20; clone DRP3/20C5, IgG1; Vector Laboratories, Burlingame, CA). β-Dystroglycan was revealed with NCL-b-DG (1:50; Novocastra). α-Sarcoglycan was detected with VP-A105 (1:50; Vector Laboratories). β-Sarcoglycan was revealed with NCL-b-SARC (1:50; Novocastra). γ-Sarcoglycan was detected with VP-G803 (1:50; Vector Laboratories). δ-Sarcoglycan was detected with sc28281 (1:50; Santa Cruz Biotechnology Inc.).

    Techniques: Double Knockout, Mouse Assay

    Rac1 regulated exercise‐induced GLUT4 translocation A , representative images showing cryosections of tibialis anterior muscle (cross sections) from basal and exercise‐stimulated Rac1 mKO and WT littermate mice stained with antibodies against GLUT4 (green) and α‐sarcoglycan (red). Scale bar = 50 μm. B , illustration of the quantification approach. Red arrows indicate the locations of the lines across and perpendicular to the plasma membrane of which 5 μm were analysed and quantified. C , quantification of the intensity of plasma membrane GLUT4 relative to α‐sarcoglycan in the basal and exercise‐stimulated (65% of individual maximal running speed, 20 min) state of WT and Rac1 mKO tibialis anterior muscle ( n = 4–6). D , 2‐DG uptake in basal and exercise‐stimulated (65% of individual maximum running speed, 20 min) tibialis anterior muscle ( n = 5 or 6). E , correlation between plasma membrane GLUT4 (GLUT4/α‐sarcoglycan) and 2‐DG uptake in basal and exercise‐stimulated tibialis anterior muscle. Included are only samples analysed for both parameters in the same muscle ( n = 4 or 5). Significant difference between basal and exercise‐stimulated intensity of GLUT4/α‐sarcoglycan or 2‐DG uptake is indicated: * P

    Journal: The Journal of Physiology

    Article Title: Rac1 governs exercise‐stimulated glucose uptake in skeletal muscle through regulation of GLUT4 translocation in mice

    doi: 10.1113/JP272039

    Figure Lengend Snippet: Rac1 regulated exercise‐induced GLUT4 translocation A , representative images showing cryosections of tibialis anterior muscle (cross sections) from basal and exercise‐stimulated Rac1 mKO and WT littermate mice stained with antibodies against GLUT4 (green) and α‐sarcoglycan (red). Scale bar = 50 μm. B , illustration of the quantification approach. Red arrows indicate the locations of the lines across and perpendicular to the plasma membrane of which 5 μm were analysed and quantified. C , quantification of the intensity of plasma membrane GLUT4 relative to α‐sarcoglycan in the basal and exercise‐stimulated (65% of individual maximal running speed, 20 min) state of WT and Rac1 mKO tibialis anterior muscle ( n = 4–6). D , 2‐DG uptake in basal and exercise‐stimulated (65% of individual maximum running speed, 20 min) tibialis anterior muscle ( n = 5 or 6). E , correlation between plasma membrane GLUT4 (GLUT4/α‐sarcoglycan) and 2‐DG uptake in basal and exercise‐stimulated tibialis anterior muscle. Included are only samples analysed for both parameters in the same muscle ( n = 4 or 5). Significant difference between basal and exercise‐stimulated intensity of GLUT4/α‐sarcoglycan or 2‐DG uptake is indicated: * P

    Article Snippet: Cryosections were fixed for 30 min in ice‐cold 4% Zamboni buffer (4% paraformaldehyde, 0.15% picric acid, 0.1 m Sorensen's phosphate buffer, pH 7.3) and washed 3 × 10 min with phosphate‐buffered saline (PBS) and incubated for 2 h with primary antibodies raised against GLUT4 (rabbit polyclonal; #PA523052; Thermo Scientific, Waltham, MA, USA) and α‐sarcoglycan (mouse monoclonal, #IVD3(1)A9; Developmental Studies Hybridoma Bank, Iowa City, IA, USA).

    Techniques: Translocation Assay, Mouse Assay, Staining

    Differences in α-dystrobrevin splice variants between cardiac and skeletal muscle DAPC. ( A ) Western blot analysis of α-dystrobrevins in mouse cardiac (C) and skeletal (S) muscle total protein lysates, DYS-IPs and IgG-IPs. α3-dystrobrevin associates with dystrophin in the heart. Fold differences in α-dystrobrevin abundance in cardiac vs. skeletal muscle DYS-IPs relative to dystrophin are shown (averages ±SD, N = 3). ( B ) Immunolabeling of wild type cardiac sections for α1 and α2-dystrobrevins. Scale bar: 50 µm. ( C ) Immunolabeling of mdx cardiac tissue section for β-sarcoglycan. Scale bar: 50 µm. ( D ) Western blot analysis of α-dystrobrevins in heart protein lysates from wild type (WT) and mdx mice. Fold differences in α-dystrobrevin abundance in WT vs. mdx cardiac lysates relative to GAPDH are shown (averages ±SD, N = 3) ( E ) Western blot analysis of α-dystrobrevins in DYS-IPs from human (H) and mouse (M) cardiac samples. Additional α-dystrobrevin isoforms (arrow heads) are detected in human cardiac lysates and DYS-IP.

    Journal: PLoS ONE

    Article Title: Proteomic Analysis Reveals New Cardiac-Specific Dystrophin-Associated Proteins

    doi: 10.1371/journal.pone.0043515

    Figure Lengend Snippet: Differences in α-dystrobrevin splice variants between cardiac and skeletal muscle DAPC. ( A ) Western blot analysis of α-dystrobrevins in mouse cardiac (C) and skeletal (S) muscle total protein lysates, DYS-IPs and IgG-IPs. α3-dystrobrevin associates with dystrophin in the heart. Fold differences in α-dystrobrevin abundance in cardiac vs. skeletal muscle DYS-IPs relative to dystrophin are shown (averages ±SD, N = 3). ( B ) Immunolabeling of wild type cardiac sections for α1 and α2-dystrobrevins. Scale bar: 50 µm. ( C ) Immunolabeling of mdx cardiac tissue section for β-sarcoglycan. Scale bar: 50 µm. ( D ) Western blot analysis of α-dystrobrevins in heart protein lysates from wild type (WT) and mdx mice. Fold differences in α-dystrobrevin abundance in WT vs. mdx cardiac lysates relative to GAPDH are shown (averages ±SD, N = 3) ( E ) Western blot analysis of α-dystrobrevins in DYS-IPs from human (H) and mouse (M) cardiac samples. Additional α-dystrobrevin isoforms (arrow heads) are detected in human cardiac lysates and DYS-IP.

    Article Snippet: Antibodies to DAPC members are: isoform specific anti- α1-, β1- or β2-syntrophin, and anti- α1- or α2-dystrobrevin antibodies , , ; pan anti-syntrophin (ab11425, Abcam); Manex1011B to dystrophin and MANDAG2 to β-dystroglycan (DSHB); clone IIH6C4 to α-dystroglycan (Upstate); anti-nNOS (#610308) and anti-α-dystrobrevin (#610766, BD Bioscience); anti-β-sarcoglycan (clone 5B1, Leica Microsystems).

    Techniques: Western Blot, Immunolabeling, Mouse Assay

    Dependence of ecto-nucleotidase activity of α-sarcoglycan-expressing HEK-293 cells on the bivalent cation concentrations ( A ) ATP-hydrolysing activity was measured in stably transfected cells in the presence of 4 mM Mg 2+ or 2 mM Ca 2+ or both (4 mM Mg 2+ and 2 mM Ca 2+ ). ( B ) ATP-hydrolysing activity of α-sarcoglycan stably transfected HEK-293 cells was measured either in the presence of 4 mM Mg 2+ and the indicated concentrations of Ca 2+ (○) or in the presence of 2 mM Ca 2+ and the indicated concentrations of Mg 2+ (•). The plot shows the values after the subtraction of the empty vector-transfected activity. Results are from four experiments performed in triplicate. ** P

    Journal: Biochemical Journal

    Article Title: Characterization of the ATP-hydrolysing activity of ?-sarcoglycan

    doi: 10.1042/BJ20031644

    Figure Lengend Snippet: Dependence of ecto-nucleotidase activity of α-sarcoglycan-expressing HEK-293 cells on the bivalent cation concentrations ( A ) ATP-hydrolysing activity was measured in stably transfected cells in the presence of 4 mM Mg 2+ or 2 mM Ca 2+ or both (4 mM Mg 2+ and 2 mM Ca 2+ ). ( B ) ATP-hydrolysing activity of α-sarcoglycan stably transfected HEK-293 cells was measured either in the presence of 4 mM Mg 2+ and the indicated concentrations of Ca 2+ (○) or in the presence of 2 mM Ca 2+ and the indicated concentrations of Mg 2+ (•). The plot shows the values after the subtraction of the empty vector-transfected activity. Results are from four experiments performed in triplicate. ** P

    Article Snippet: The proteins (40–50 μg/lane) were resolved by SDS/PAGE, blotted on to a nitrocellulose membrane and examined with the following antibodies: mouse monoclonal anti-α-sarcoglycan (NCL-a-SARC, 1:500), anti-dystrophin (NCL-DYS2, 1:100), and anti-β-dystroglycan (NCL-b-DG, 1:100) from Novocastra and rabbit polyclonal anti-β-actin (AC-15, 1:4000) from Sigma.

    Techniques: Activity Assay, Expressing, Stable Transfection, Transfection, Plasmid Preparation

    Transient transfection of HEK-293 cells with α-sarcoglycan ( A ) Western-blot analysis of rabbit skeletal-muscle sarcolemma (rabbit SL), and HEK-293 cell lysates from α-sarcoglycan-transiently transfected cells (α-SG), empty vector (pcDNA3) transfected cells and untransfected cells (wild-type) by using an antibody specific to α-sarcoglycan. ( B ) Western-blot analysis of rabbit skeletal-muscle sarcolemma (rabbit SL), and HEK-293 cell lysates from α-sarcoglycan-transiently transfected cells (HEK α-SG) either untreated (left two lanes) or treated with N -glycosidase F (right two lanes). Amounts of protein from HEK-293 cell lysates were estimated ( A , B ) by using an antibody against β-actin. ( C ) Phase-contrast image (left panel) and immunofluorescence staining of non-permeabilized α-sarcoglycan transiently transfected HEK-293 cells with the antibody directed against the extracellular domain of α-sarcoglycan (right panel). ( D ) Extracellular ATP hydrolysis of wild-type HEK-293 cells (wt), or cells transiently transfected with the empty vector (pcDNA3) or with α-sarcoglycan (α-SG). Results are from three experiments performed in triplicate; ** P

    Journal: Biochemical Journal

    Article Title: Characterization of the ATP-hydrolysing activity of ?-sarcoglycan

    doi: 10.1042/BJ20031644

    Figure Lengend Snippet: Transient transfection of HEK-293 cells with α-sarcoglycan ( A ) Western-blot analysis of rabbit skeletal-muscle sarcolemma (rabbit SL), and HEK-293 cell lysates from α-sarcoglycan-transiently transfected cells (α-SG), empty vector (pcDNA3) transfected cells and untransfected cells (wild-type) by using an antibody specific to α-sarcoglycan. ( B ) Western-blot analysis of rabbit skeletal-muscle sarcolemma (rabbit SL), and HEK-293 cell lysates from α-sarcoglycan-transiently transfected cells (HEK α-SG) either untreated (left two lanes) or treated with N -glycosidase F (right two lanes). Amounts of protein from HEK-293 cell lysates were estimated ( A , B ) by using an antibody against β-actin. ( C ) Phase-contrast image (left panel) and immunofluorescence staining of non-permeabilized α-sarcoglycan transiently transfected HEK-293 cells with the antibody directed against the extracellular domain of α-sarcoglycan (right panel). ( D ) Extracellular ATP hydrolysis of wild-type HEK-293 cells (wt), or cells transiently transfected with the empty vector (pcDNA3) or with α-sarcoglycan (α-SG). Results are from three experiments performed in triplicate; ** P

    Article Snippet: The proteins (40–50 μg/lane) were resolved by SDS/PAGE, blotted on to a nitrocellulose membrane and examined with the following antibodies: mouse monoclonal anti-α-sarcoglycan (NCL-a-SARC, 1:500), anti-dystrophin (NCL-DYS2, 1:100), and anti-β-dystroglycan (NCL-b-DG, 1:100) from Novocastra and rabbit polyclonal anti-β-actin (AC-15, 1:4000) from Sigma.

    Techniques: Transfection, Western Blot, Plasmid Preparation, Immunofluorescence, Staining

    Ecto-nucleotidase activity of HEK-293 cells stably transfected with α-sarcoglycan ( A ) ATP-hydrolysing activity, measured as indicated in the Experimental section, was performed in HEK-293 cells transfected with the empty vector (pcDNA3) or with α-sarcoglycan (α-SG). Results are from four experiments performed in triplicate; ** P

    Journal: Biochemical Journal

    Article Title: Characterization of the ATP-hydrolysing activity of ?-sarcoglycan

    doi: 10.1042/BJ20031644

    Figure Lengend Snippet: Ecto-nucleotidase activity of HEK-293 cells stably transfected with α-sarcoglycan ( A ) ATP-hydrolysing activity, measured as indicated in the Experimental section, was performed in HEK-293 cells transfected with the empty vector (pcDNA3) or with α-sarcoglycan (α-SG). Results are from four experiments performed in triplicate; ** P

    Article Snippet: The proteins (40–50 μg/lane) were resolved by SDS/PAGE, blotted on to a nitrocellulose membrane and examined with the following antibodies: mouse monoclonal anti-α-sarcoglycan (NCL-a-SARC, 1:500), anti-dystrophin (NCL-DYS2, 1:100), and anti-β-dystroglycan (NCL-b-DG, 1:100) from Novocastra and rabbit polyclonal anti-β-actin (AC-15, 1:4000) from Sigma.

    Techniques: Activity Assay, Stable Transfection, Transfection, Plasmid Preparation

    Effects of antibodies against α-sarcoglycan on the ecto-nucleotidase activity of HEK-293 cells stably expressing the protein ATP-hydrolysing activity of cells incubated in the absence or in the presence of a monoclonal antibody against the extracellular domain of α-sarcoglycan encompassing the ATP-binding site (+mAb) or a polyclonal antibody specific for the C-terminal domain of the protein (+pAb). HEK-293 cells were preincubated for 30 min at 4 °C with or without antibodies. Results are from four experiments performed in triplicate; ** P

    Journal: Biochemical Journal

    Article Title: Characterization of the ATP-hydrolysing activity of ?-sarcoglycan

    doi: 10.1042/BJ20031644

    Figure Lengend Snippet: Effects of antibodies against α-sarcoglycan on the ecto-nucleotidase activity of HEK-293 cells stably expressing the protein ATP-hydrolysing activity of cells incubated in the absence or in the presence of a monoclonal antibody against the extracellular domain of α-sarcoglycan encompassing the ATP-binding site (+mAb) or a polyclonal antibody specific for the C-terminal domain of the protein (+pAb). HEK-293 cells were preincubated for 30 min at 4 °C with or without antibodies. Results are from four experiments performed in triplicate; ** P

    Article Snippet: The proteins (40–50 μg/lane) were resolved by SDS/PAGE, blotted on to a nitrocellulose membrane and examined with the following antibodies: mouse monoclonal anti-α-sarcoglycan (NCL-a-SARC, 1:500), anti-dystrophin (NCL-DYS2, 1:100), and anti-β-dystroglycan (NCL-b-DG, 1:100) from Novocastra and rabbit polyclonal anti-β-actin (AC-15, 1:4000) from Sigma.

    Techniques: Activity Assay, Stable Transfection, Expressing, Incubation, Binding Assay

    Western-blot analysis and total ecto-nucleotidase activity of C2C12 cells ( A ) Western-blot analysis of cell lysates obtained from proliferating (P), confluent (C) and myotubes at different days of culture. Lysate proteins (50 μg) were separated on 10% SDS/polyacrylamide slab gels, blotted on nitrocellulose filters and probed with specific antibodies, as indicated. ( B ) ATP-hydrolysing activity of C2C12 myoblasts (Mb) and 4- and 8-day-old myotubes (4d-Mt and 8d-Mt respectively) was determined by measuring P i ]. Cells were incubated for 15 min in a medium with the following composition: 20 mM Hepes, 140 mM NaCl, 4 mM MgCl 2 and 2 mM CaCl 2 . The reaction was started by adding 4 mM ATP and terminated by withdrawing a supernatant aliquot. Results are from four experiments performed in triplicate. ( C ) Effect of polyclonal (+pAb) and monoclonal (+mAb) antibodies specific for α-sarcoglycan on total ecto-ATPase activity of 7-day-old C2C12 myotubes incubated 30 min as above. Results are from three experiments performed in triplicate. * P

    Journal: Biochemical Journal

    Article Title: Characterization of the ATP-hydrolysing activity of ?-sarcoglycan

    doi: 10.1042/BJ20031644

    Figure Lengend Snippet: Western-blot analysis and total ecto-nucleotidase activity of C2C12 cells ( A ) Western-blot analysis of cell lysates obtained from proliferating (P), confluent (C) and myotubes at different days of culture. Lysate proteins (50 μg) were separated on 10% SDS/polyacrylamide slab gels, blotted on nitrocellulose filters and probed with specific antibodies, as indicated. ( B ) ATP-hydrolysing activity of C2C12 myoblasts (Mb) and 4- and 8-day-old myotubes (4d-Mt and 8d-Mt respectively) was determined by measuring P i ]. Cells were incubated for 15 min in a medium with the following composition: 20 mM Hepes, 140 mM NaCl, 4 mM MgCl 2 and 2 mM CaCl 2 . The reaction was started by adding 4 mM ATP and terminated by withdrawing a supernatant aliquot. Results are from four experiments performed in triplicate. ( C ) Effect of polyclonal (+pAb) and monoclonal (+mAb) antibodies specific for α-sarcoglycan on total ecto-ATPase activity of 7-day-old C2C12 myotubes incubated 30 min as above. Results are from three experiments performed in triplicate. * P

    Article Snippet: The proteins (40–50 μg/lane) were resolved by SDS/PAGE, blotted on to a nitrocellulose membrane and examined with the following antibodies: mouse monoclonal anti-α-sarcoglycan (NCL-a-SARC, 1:500), anti-dystrophin (NCL-DYS2, 1:100), and anti-β-dystroglycan (NCL-b-DG, 1:100) from Novocastra and rabbit polyclonal anti-β-actin (AC-15, 1:4000) from Sigma.

    Techniques: Western Blot, Activity Assay, Incubation

    The expression analyses of α-, β-, γ-sarcoglycan and dysferlin protein in the muscles of patient by Western blot. The total proteins extracted from skeletal muscles of normal person (lane 1), relative normal muscle biopsies of patient IV-7 (lane 2), and dystrophic muscle biopsies of patient IV-7 (lane 3).

    Journal: Journal of Translational Medicine

    Article Title: Differential expression profiling between the relative normal and dystrophic muscle tissues from the same LGMD patient

    doi: 10.1186/1479-5876-4-53

    Figure Lengend Snippet: The expression analyses of α-, β-, γ-sarcoglycan and dysferlin protein in the muscles of patient by Western blot. The total proteins extracted from skeletal muscles of normal person (lane 1), relative normal muscle biopsies of patient IV-7 (lane 2), and dystrophic muscle biopsies of patient IV-7 (lane 3).

    Article Snippet: Antibodies and Western blot Antibodies against Dysferlin (E20), α-Sarcoglycan (D-20), β-Sarcoglycan (A-17), γ-Sarcoglycan (D-18), α-Tubulin, and horseradish peroxidase (HRP)-conjugated second antibodies were from Santa Cruz Biotechnology.

    Techniques: Expressing, Western Blot

    SGCG reading frame correction in urine-derived cells (UDCs). UDCs from a normal control subject and an LGMD 2C patient with a deletion of exon 6 (ex6del) were reprogrammed into a myogenic lineage. ( A ) γ-Sarcoglycan protein (green) was detected by IFM in the reprogrammed normal control myotubes but not in reprogrammed ex6del cells. α-Actinin, red; nuclei, blue. ( B ) RT-PCR analysis demonstrated reading frame–corrected Mini-Gamma transcript expression (red arrowhead). ( C ) Representative IFM images showed the restoration of γ-sarcoglycan protein expression in cells treated with Mini-Gamma vivo-PMOs. ( D ) Significant increase in γ-sarcoglycan protein fluorescence was observed after treatment with vivo-PMOs ( n = 5) as compared with nontargeting control vivo-PMOs ( n = 5). A minimum of 3 independent fields were analyzed for each sample. ( E ) To assess membrane stability in response to vivo-PMO treatment, reprogrammed cells were challenged with hypo-osmotic shock and membrane leak was monitored by release of creatine kinase (CK). Vivo-PMO treatment significantly decreased the relative amount of CK release consistent with increased membrane stability. Data represent the percent of CK released relative to the total CK from 4 independent experiments ( n = 3–4, for each). Data are presented as the mean CK released in cells treated with exon-skipping vivo-PMOs relative to the mean in cells treated with a nontargeting vivo-PMO. ( F ) Model depicting the increased membrane stability that resulted from vivo-PMO–mediated reading frame correction of an SGCG frameshift mutation. * P

    Journal: JCI Insight

    Article Title: Efficient exon skipping of SGCG mutations mediated by phosphorodiamidate morpholino oligomers

    doi: 10.1172/jci.insight.99357

    Figure Lengend Snippet: SGCG reading frame correction in urine-derived cells (UDCs). UDCs from a normal control subject and an LGMD 2C patient with a deletion of exon 6 (ex6del) were reprogrammed into a myogenic lineage. ( A ) γ-Sarcoglycan protein (green) was detected by IFM in the reprogrammed normal control myotubes but not in reprogrammed ex6del cells. α-Actinin, red; nuclei, blue. ( B ) RT-PCR analysis demonstrated reading frame–corrected Mini-Gamma transcript expression (red arrowhead). ( C ) Representative IFM images showed the restoration of γ-sarcoglycan protein expression in cells treated with Mini-Gamma vivo-PMOs. ( D ) Significant increase in γ-sarcoglycan protein fluorescence was observed after treatment with vivo-PMOs ( n = 5) as compared with nontargeting control vivo-PMOs ( n = 5). A minimum of 3 independent fields were analyzed for each sample. ( E ) To assess membrane stability in response to vivo-PMO treatment, reprogrammed cells were challenged with hypo-osmotic shock and membrane leak was monitored by release of creatine kinase (CK). Vivo-PMO treatment significantly decreased the relative amount of CK release consistent with increased membrane stability. Data represent the percent of CK released relative to the total CK from 4 independent experiments ( n = 3–4, for each). Data are presented as the mean CK released in cells treated with exon-skipping vivo-PMOs relative to the mean in cells treated with a nontargeting vivo-PMO. ( F ) Model depicting the increased membrane stability that resulted from vivo-PMO–mediated reading frame correction of an SGCG frameshift mutation. * P

    Article Snippet: Primary antibodies were as follows: rabbit polyclonal anti–γ-sarcoglycan (1:1,000) ( ); rabbit polyclonal anti–γ-sarcoglycan (1:1,000, Abnova; MaxPab H00006445-D01); and mouse monoclonal anti–γ-tubulin (1:1,000, MilliporeSigma, T5326/GTU-88).

    Techniques: Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Fluorescence, Mutagenesis

    Restoration of SGCG expression as Mini-Gamma protein. Reprogrammed fibroblasts were treated for 3 days with either a nontargeting control vivo-PMO (nontarget) or a mutation-specific vivo-PMO exon skipping cocktail (Mini-Gamma). ( A ) Representative IFM images of reprogrammed 521ΔT cells that were treated as indicated. Treatment with reading frame–correcting vivo-PMOs resulted in the expression of the Mini-Gamma protein (green). Myotubes were labeled with α-actinin (red), and nuclei were labeled with Hoechst 3342 (blue). ( B ) Image analysis demonstrated a significant increase in Mini-Gamma fluorescence after treatment with vivo-PMOs that corrected the transcript reading frame ( n = 6) as compared with nontargeting controls ( n = 5). ( C and D ) Reprogrammed cells harboring a deletion of SGCG exons 5 and 6 (ex5/6del) were treated as indicated. IFM image analysis demonstrated a significant increase in Mini-Gamma fluorescence after treatment with vivo-PMOs that corrected the reading transcript reading frame ( n = 4) as compared with nontargeting controls ( n = 3). ( E and F ) Reprogrammed cells harboring a deletion of SGCG exons 6 (ex6del) were treated as indicated. IFM image analysis demonstrated a significant increase in Mini-Gamma fluorescence after treatment with vivo-PMOs that corrected the transcript reading frame ( n = 5) as compared with nontargeting controls ( n = 3). Data represent the mean γ-sarcoglycan fluorescence per α-actinin–positive area normalized to the mean of the untreated group. A minimum of 3 independent fields were analyzed for each sample. * P

    Journal: JCI Insight

    Article Title: Efficient exon skipping of SGCG mutations mediated by phosphorodiamidate morpholino oligomers

    doi: 10.1172/jci.insight.99357

    Figure Lengend Snippet: Restoration of SGCG expression as Mini-Gamma protein. Reprogrammed fibroblasts were treated for 3 days with either a nontargeting control vivo-PMO (nontarget) or a mutation-specific vivo-PMO exon skipping cocktail (Mini-Gamma). ( A ) Representative IFM images of reprogrammed 521ΔT cells that were treated as indicated. Treatment with reading frame–correcting vivo-PMOs resulted in the expression of the Mini-Gamma protein (green). Myotubes were labeled with α-actinin (red), and nuclei were labeled with Hoechst 3342 (blue). ( B ) Image analysis demonstrated a significant increase in Mini-Gamma fluorescence after treatment with vivo-PMOs that corrected the transcript reading frame ( n = 6) as compared with nontargeting controls ( n = 5). ( C and D ) Reprogrammed cells harboring a deletion of SGCG exons 5 and 6 (ex5/6del) were treated as indicated. IFM image analysis demonstrated a significant increase in Mini-Gamma fluorescence after treatment with vivo-PMOs that corrected the reading transcript reading frame ( n = 4) as compared with nontargeting controls ( n = 3). ( E and F ) Reprogrammed cells harboring a deletion of SGCG exons 6 (ex6del) were treated as indicated. IFM image analysis demonstrated a significant increase in Mini-Gamma fluorescence after treatment with vivo-PMOs that corrected the transcript reading frame ( n = 5) as compared with nontargeting controls ( n = 3). Data represent the mean γ-sarcoglycan fluorescence per α-actinin–positive area normalized to the mean of the untreated group. A minimum of 3 independent fields were analyzed for each sample. * P

    Article Snippet: Primary antibodies were as follows: rabbit polyclonal anti–γ-sarcoglycan (1:1,000) ( ); rabbit polyclonal anti–γ-sarcoglycan (1:1,000, Abnova; MaxPab H00006445-D01); and mouse monoclonal anti–γ-tubulin (1:1,000, MilliporeSigma, T5326/GTU-88).

    Techniques: Expressing, Mutagenesis, Labeling, Fluorescence

    An exon-skipping strategy to treat LGMD 2C. ( A ) The SGCG gene, which encodes γ-sarcoglycan, is comprised of 8 exons, and many SGCG mutations disrupt the transcript reading frame, causing Limb-girdle muscular dystrophy (LGMD) 2C. ( B ) Correction of the reading frame requires the skipping of SGCG exons 4, 5, 6, and 7 at the pre-mRNA level. Four antisense oligonucleotides (AON) were designed to exclude these exons from the mature mRNA transcript. The bypass of exons 4–7 generates an internally truncated product termed Mini-Gamma encoded by exons 2, 3, and 8. ( C ). ( D ) Exon skipping AONs are chemically modified to avoid nuclease degradation and destruction of the mRNA transcript. Shown are the chemical structures of 2 types of AONs used for exon skipping. 2’- O -methyl phosphorothioates (2 O ).

    Journal: JCI Insight

    Article Title: Efficient exon skipping of SGCG mutations mediated by phosphorodiamidate morpholino oligomers

    doi: 10.1172/jci.insight.99357

    Figure Lengend Snippet: An exon-skipping strategy to treat LGMD 2C. ( A ) The SGCG gene, which encodes γ-sarcoglycan, is comprised of 8 exons, and many SGCG mutations disrupt the transcript reading frame, causing Limb-girdle muscular dystrophy (LGMD) 2C. ( B ) Correction of the reading frame requires the skipping of SGCG exons 4, 5, 6, and 7 at the pre-mRNA level. Four antisense oligonucleotides (AON) were designed to exclude these exons from the mature mRNA transcript. The bypass of exons 4–7 generates an internally truncated product termed Mini-Gamma encoded by exons 2, 3, and 8. ( C ). ( D ) Exon skipping AONs are chemically modified to avoid nuclease degradation and destruction of the mRNA transcript. Shown are the chemical structures of 2 types of AONs used for exon skipping. 2’- O -methyl phosphorothioates (2 O ).

    Article Snippet: Primary antibodies were as follows: rabbit polyclonal anti–γ-sarcoglycan (1:1,000) ( ); rabbit polyclonal anti–γ-sarcoglycan (1:1,000, Abnova; MaxPab H00006445-D01); and mouse monoclonal anti–γ-tubulin (1:1,000, MilliporeSigma, T5326/GTU-88).

    Techniques: Modification