alpha-sarcoglycan Search Results


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  • 85
    Leica Biosystems beta sarcoglycan
    Immunostaining of quadriceps muscle samples for Patients 1 and 2. Complete absence of staining (K and M) for  γ -sarcoglycan antibody is demonstrated in both patients. Patient 1′s muscle tissue (E, H, and N) shows partial reduction in staining of  α -,  β -, and  δ -sarcoglycan, while tissue on Patient 2 (F, I, and O) demonstrates loss of expression of  α - and  β -sarcoglycan and reduction in  δ -sarcoglycan.
    Beta Sarcoglycan, supplied by Leica Biosystems, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    MONOSAN anti alpha sarcoglycan
    Isolation of <t>alpha-sarcoglycan</t> + EVs from plasma using immunoaffinity capturing. Anti-alpha-sarcoglycan antibodies were conjugated to magnetic beads to isolate muscle EVs from plasma. Western blot analysis confirmed the presence of the exosomal marker Tsg101 in isolated EVs. Ponceau S Staining has been used as loading control (a). MicroRNA quantifications showed an increase in the miR-206/miR-16 ratio in the SGCA + sub-population of EVs (SGCA-Beads) compared to total (total plasma EVs) or uncaptured EVs (Supernatant). MiR-206 expression levels were normalized versus the endogenous reference miR-16 and expressed as -ΔCq (where ΔCq = Cq miR-206 -Cq miR-16 ) (b). Moreover, the quantification of miR-16 ratio in the SGCA + sub-population of EVs compared to total or uncaptured EVs shows that SGCA-conjugated beads retained about 2–5% of the total amount of EVs, miR-16 expression levels were normalized versus the spike-in reference cel-miR-39 and expressed as -ΔCq (where ΔCq = Cq miR-16 -Cq cel-miR-39 ) (c). Asterisks denote significant changes ( p
    Anti Alpha Sarcoglycan, supplied by MONOSAN, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology alpha sarcoglycan
    Isolation of <t>alpha-sarcoglycan</t> + EVs from plasma using immunoaffinity capturing. Anti-alpha-sarcoglycan antibodies were conjugated to magnetic beads to isolate muscle EVs from plasma. Western blot analysis confirmed the presence of the exosomal marker Tsg101 in isolated EVs. Ponceau S Staining has been used as loading control (a). MicroRNA quantifications showed an increase in the miR-206/miR-16 ratio in the SGCA + sub-population of EVs (SGCA-Beads) compared to total (total plasma EVs) or uncaptured EVs (Supernatant). MiR-206 expression levels were normalized versus the endogenous reference miR-16 and expressed as -ΔCq (where ΔCq = Cq miR-206 -Cq miR-16 ) (b). Moreover, the quantification of miR-16 ratio in the SGCA + sub-population of EVs compared to total or uncaptured EVs shows that SGCA-conjugated beads retained about 2–5% of the total amount of EVs, miR-16 expression levels were normalized versus the spike-in reference cel-miR-39 and expressed as -ΔCq (where ΔCq = Cq miR-16 -Cq cel-miR-39 ) (c). Asterisks denote significant changes ( p
    Alpha 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
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    sgα  (Abcam)
    99
    Abcam sgα
    Ameliorated NMJ fragmentation and denervation in aged mice by <t>SGα</t> overexpression. A , Reduced fragmentation and improved innervation. Shown were TA muscles of mice at different ages and indicated AAV treatment, whole-mount stained with CF568 α-BTX (red) and anti-NF/Syn antibodies (visualized by AlexaFluor 488 goat anti-rabbit IgG; green). Blue arrowhead, denervated or partially innervated endplate. Scale bar, 50 μm. B , Enlarged images of individual endplates. Scale bar, 10 μm. C – F , Quantitative analysis of data in A . C , Rescue of denervation in aged mice by SGα expression; F (2,12) = 29, *** p = 0.0001, ** p = 0.0051. D , Rescue of fragmentation number in aged mice by SGα expression; F (2,12) = 25.8, *** p = 0.0001, ** p = 0.0023. E , Rescue of fragmented endplate percentage in aged mice by SGα expression; F (2,12) = 93, *** p = 0.0001, *** p = 0.0003. F , Rescue of reduced AChR cluster intensity in aged mice by SGα expression; F (2,12) = 35.9, *** p = 0.0001, ** p = 0.0028; N = 5 mice per group, one-way ANOVA.
    Sgα, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MONOSAN biotinylated anti alpha sarcoglycan
    Isolation of <t>alpha-sarcoglycan</t> + EVs from plasma using immunoaffinity capturing. Anti-alpha-sarcoglycan antibodies were conjugated to magnetic beads to isolate muscle EVs from plasma. Western blot analysis confirmed the presence of the exosomal marker Tsg101 in isolated EVs. Ponceau S Staining has been used as loading control (a). MicroRNA quantifications showed an increase in the miR-206/miR-16 ratio in the SGCA + sub-population of EVs (SGCA-Beads) compared to total (total plasma EVs) or uncaptured EVs (Supernatant). MiR-206 expression levels were normalized versus the endogenous reference miR-16 and expressed as -ΔCq (where ΔCq = Cq miR-206 -Cq miR-16 ) (b). Moreover, the quantification of miR-16 ratio in the SGCA + sub-population of EVs compared to total or uncaptured EVs shows that SGCA-conjugated beads retained about 2–5% of the total amount of EVs, miR-16 expression levels were normalized versus the spike-in reference cel-miR-39 and expressed as -ΔCq (where ΔCq = Cq miR-16 -Cq cel-miR-39 ) (c). Asterisks denote significant changes ( p
    Biotinylated Anti Alpha Sarcoglycan, supplied by MONOSAN, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Novocastra adhalin
    The muscle biopsy of the patient with the genetically confirmed LGMD2D showing no sign of dystrophy (A) and normal expression of all <t>sarcoglycans</t> (B, C, D, E) . The biopsy was taken at the age of 8 years.
    Adhalin, supplied by Novocastra, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Millipore alpha sarcoglycan
    The muscle biopsy of the patient with the genetically confirmed LGMD2D showing no sign of dystrophy (A) and normal expression of all <t>sarcoglycans</t> (B, C, D, E) . The biopsy was taken at the age of 8 years.
    Alpha Sarcoglycan, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Journal of Biological Chemistry sarcoglycan adhalin
    The muscle biopsy of the patient with the genetically confirmed LGMD2D showing no sign of dystrophy (A) and normal expression of all <t>sarcoglycans</t> (B, C, D, E) . The biopsy was taken at the age of 8 years.
    Sarcoglycan Adhalin, supplied by Journal of Biological Chemistry, used in various techniques. Bioz Stars score: 84/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Novocastra α sarcoglycan adhalin
    The muscle biopsy of the patient with the genetically confirmed LGMD2D showing no sign of dystrophy (A) and normal expression of all <t>sarcoglycans</t> (B, C, D, E) . The biopsy was taken at the age of 8 years.
    α Sarcoglycan Adhalin, supplied by Novocastra, used in various techniques. Bioz Stars score: 81/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher alpha sarcoglycan immunoaffinity capture beads exosome dynabeads streptavidin
    The muscle biopsy of the patient with the genetically confirmed LGMD2D showing no sign of dystrophy (A) and normal expression of all <t>sarcoglycans</t> (B, C, D, E) . The biopsy was taken at the age of 8 years.
    Alpha Sarcoglycan Immunoaffinity Capture Beads Exosome Dynabeads Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Novocastra gamma sarcoglycans
    The muscle biopsy of the patient with the genetically confirmed LGMD2D showing no sign of dystrophy (A) and normal expression of all <t>sarcoglycans</t> (B, C, D, E) . The biopsy was taken at the age of 8 years.
    Gamma Sarcoglycans, supplied by Novocastra, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Novus Biologicals beta sarcoglycan antibody
    The muscle biopsy of the patient with the genetically confirmed LGMD2D showing no sign of dystrophy (A) and normal expression of all <t>sarcoglycans</t> (B, C, D, E) . The biopsy was taken at the age of 8 years.
    Beta Sarcoglycan Antibody, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Novocastra anti β sarcoglycans
    Dantrolene/e23AON Combination Therapy Restores Sarcolemmal Dystrophin and DGC after Chronic Treatment Effect of dantrolene/e23AON combination therapy on rescue of DGC components as illustrated by representative immunofluorescence images of serial cross-sections from treated mdx quadriceps. Dystrophin was detected with an antibody specific to the rod domain (MANDYS8). Additional DGC components, <t>α-sarcoglycan</t> and β-dystroglycan, were detected with NCL-α-SARC and NCL-β-DG antibodies, respectively. ‡ Dantrolene was dosed at 30–70 mg/kg/day.
    Anti β Sarcoglycans, supplied by Novocastra, used in various techniques. Bioz Stars score: 92/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Novocastra δ sarcoglycans
    Dantrolene/e23AON Combination Therapy Restores Sarcolemmal Dystrophin and DGC after Chronic Treatment Effect of dantrolene/e23AON combination therapy on rescue of DGC components as illustrated by representative immunofluorescence images of serial cross-sections from treated mdx quadriceps. Dystrophin was detected with an antibody specific to the rod domain (MANDYS8). Additional DGC components, <t>α-sarcoglycan</t> and β-dystroglycan, were detected with NCL-α-SARC and NCL-β-DG antibodies, respectively. ‡ Dantrolene was dosed at 30–70 mg/kg/day.
    δ Sarcoglycans, supplied by Novocastra, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GeneTex anti delta sarcoglycan
    Dantrolene/e23AON Combination Therapy Restores Sarcolemmal Dystrophin and DGC after Chronic Treatment Effect of dantrolene/e23AON combination therapy on rescue of DGC components as illustrated by representative immunofluorescence images of serial cross-sections from treated mdx quadriceps. Dystrophin was detected with an antibody specific to the rod domain (MANDYS8). Additional DGC components, <t>α-sarcoglycan</t> and β-dystroglycan, were detected with NCL-α-SARC and NCL-β-DG antibodies, respectively. ‡ Dantrolene was dosed at 30–70 mg/kg/day.
    Anti Delta Sarcoglycan, supplied by GeneTex, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Developmental Studies Hybridoma Bank sarcoglycans α
    Dantrolene/e23AON Combination Therapy Restores Sarcolemmal Dystrophin and DGC after Chronic Treatment Effect of dantrolene/e23AON combination therapy on rescue of DGC components as illustrated by representative immunofluorescence images of serial cross-sections from treated mdx quadriceps. Dystrophin was detected with an antibody specific to the rod domain (MANDYS8). Additional DGC components, <t>α-sarcoglycan</t> and β-dystroglycan, were detected with NCL-α-SARC and NCL-β-DG antibodies, respectively. ‡ Dantrolene was dosed at 30–70 mg/kg/day.
    Sarcoglycans α, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Novocastra mouse monoclonal anti adhalin antibody
    Dantrolene/e23AON Combination Therapy Restores Sarcolemmal Dystrophin and DGC after Chronic Treatment Effect of dantrolene/e23AON combination therapy on rescue of DGC components as illustrated by representative immunofluorescence images of serial cross-sections from treated mdx quadriceps. Dystrophin was detected with an antibody specific to the rod domain (MANDYS8). Additional DGC components, <t>α-sarcoglycan</t> and β-dystroglycan, were detected with NCL-α-SARC and NCL-β-DG antibodies, respectively. ‡ Dantrolene was dosed at 30–70 mg/kg/day.
    Mouse Monoclonal Anti Adhalin Antibody, supplied by Novocastra, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Novus Biologicals β sarcoglycan
    Impaired sarcolemmal glycoprotein trafficking in hearts overexpressing secretion-defective Thbs4. (A) Western blotting for membrane glycoproteins purified from hearts of transgenic mice overexpressing wild-type Thbs4 (DTG-Thbs4) or the secretion-defective mutant (DTG-mCa 2+ ). Cadherin was used as a loading control. (B) Representative experiment of immunocytochemistry for <t>β-sarcoglycan</t> and the Golgi marker GM130 in cardiomyocytes isolated from transgenic hearts of the indicated genotype. Scale bar = 50 μm. Similar results were obtained in 2 additional experiments.
    β Sarcoglycan, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    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: 93/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Takeda takeda s epsilon 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
    Takeda S Epsilon Sarcoglycan, supplied by Takeda, used in various techniques. Bioz Stars score: 90/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam anti δ sarcoglycan leica
    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
    Anti δ Sarcoglycan Leica, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Novus Biologicals rabbit polyclonal anti β 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
    Rabbit Polyclonal Anti β Sarcoglycan, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Abcam c terminal delta sarcoglycan δ sg
    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
    C Terminal Delta Sarcoglycan δ Sg, supplied by Abcam, used in various techniques. Bioz Stars score: 84/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Leica Biosystems mouse monoclonal beta sarcoglycan antibodies
    Morphological and molecular evidences of myocardial hypertrophy progression. (A) Immunohistochemical staining of <t>beta-</t> <t>sarcoglycan</t> allowing to detect outer membrane showing clear increase of cells size in transverse orientation by 10-weeks group compared to intact. (B) Increase in cell diameter (Dmin) illustrating progressive cardiomyocytes enlargement, and significant increase after 8 and 10 weeks of aortic banding performing. Nppa expression was upregulated in LV (C) and IVS (D) after 1, 2, 8, and 10 weeks of model duration compared to intact or sham-control groups. (E) Positive linear correlation was found for Nppa mRNA level and left ventricular mass (LVM) indexed to body weight. The analysis included experimental groups after 8 and 10 weeks of aortic constriction, 8 week’s sham-operated and intact animals, r indicates Pearson coefficient; for all ∗ P
    Mouse Monoclonal Beta Sarcoglycan Antibodies, supplied by Leica Biosystems, used in various techniques. Bioz Stars score: 95/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    R&D Systems goat anti human δ sarcoglycan
    Adenoviral gene transfer efficiency and expression of wild-type (WT) and mutant <t>δ-sarcoglycan</t> (δSG) in adult cardiac myocytes. A : δSG adenoviral vector design. B : time course of δSG expression using adenoviral vectors in
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    92
    Developmental Studies Hybridoma Bank sarcoglycan complex antibodies rabbit anti α sarcoglycan
    Adenoviral gene transfer efficiency and expression of wild-type (WT) and mutant <t>δ-sarcoglycan</t> (δSG) in adult cardiac myocytes. A : δSG adenoviral vector design. B : time course of δSG expression using adenoviral vectors in
    Sarcoglycan Complex Antibodies Rabbit Anti α Sarcoglycan, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti human specific γ sarcoglycan antibody
    Adenoviral gene transfer efficiency and expression of wild-type (WT) and mutant <t>δ-sarcoglycan</t> (δSG) in adult cardiac myocytes. A : δSG adenoviral vector design. B : time course of δSG expression using adenoviral vectors in
    Anti Human Specific γ Sarcoglycan Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Novocastra β sarcoglycan
    Functional and phenotypic correction in mdx mice following treatments with PNA AOs at 100 mg/kg for five weekly intravenous injections . ( a ) Serial immunostaining of dystrophin-associated protein complex (DAPC) in mdx mice. DAPC protein components β-dystroglycan, <t>α-sarcoglycan,</t> and <t>β-sarcoglycan</t> and neuronal nitric oxide synthase (nNOS) were detected in quadriceps from mdx mice treated with PNA AOs. Arrowheads point to the identical muscle fibers (bar = 200 μm). ( b ) Muscle function was assessed using grip strength test to determine the physical improvement of PNA AOs treated mdx mice. Significant force recovery was detected in treated mdx mice compared with untreated mdx controls ( t -test, * P
    β Sarcoglycan, supplied by Novocastra, used in various techniques. Bioz Stars score: 92/100, based on 146 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Leica Biosystems novocastratm lyophilized mouse monoclonal antibody gamma sarcoglycan
    Functional and phenotypic correction in mdx mice following treatments with PNA AOs at 100 mg/kg for five weekly intravenous injections . ( a ) Serial immunostaining of dystrophin-associated protein complex (DAPC) in mdx mice. DAPC protein components β-dystroglycan, <t>α-sarcoglycan,</t> and <t>β-sarcoglycan</t> and neuronal nitric oxide synthase (nNOS) were detected in quadriceps from mdx mice treated with PNA AOs. Arrowheads point to the identical muscle fibers (bar = 200 μm). ( b ) Muscle function was assessed using grip strength test to determine the physical improvement of PNA AOs treated mdx mice. Significant force recovery was detected in treated mdx mice compared with untreated mdx controls ( t -test, * P
    Novocastratm Lyophilized Mouse Monoclonal Antibody Gamma Sarcoglycan, supplied by Leica Biosystems, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Novocastra 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 Novocastra, used in various techniques. Bioz Stars score: 86/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Novocastra γ sarcoglycan
    HDAC4 directly represses structural gene expression via binding and inhibition of MEF2 activity. A ) Promoter sequence of <t>γ-sarcoglycan</t> spanning −812 to −861 nt upstream of transcriptional start. AT-rich MEF2 sites are indicated above, which were mutated singly in or combination for subsequent luciferase assays. B ) C2C12 myoblasts were tranfected with luciferase reporter constructs containing wild-type or MEF2 binding site deletion (ΔAT1, ΔAT2, or ΔAT1,2) promoters as indicated. Transfections occurred in the presence of MEF2 and/or HDAC4 as indicated. Luciferase activity was determined 48 h after transfection. C . D ) ChIP analysis was performed on C2C12 myotubes using either control IgG-, MEF2-, or HDAC4-specific antibodies. Bound DNA was analyzed by standard PCR generating a 300 bp fragment encompassing both MEF2 binding sites in the γ-sarcoglycan promoter. Input lane represents 5% of total starting material.
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    Santa Cruz Biotechnology sarcoglycan
    β, γ, δ, and ε <t>sarcoglycan</t> are not overexpressed in Galgt2 transgenic Sgca −/− muscle. A : Immunostaining of gastrocnemius muscle in Galgt2 transgenic (CT) and non-transgenic Sgca +/− and Sgca −/− mice. β, γ, and ε sarcoglycan were not increased in expression along myofibers in Sgca −/− CT muscle, while ( B ) immunostaining of α dystroglycan, β dystroglycan, dystrophin, and utrophin was high in Sgca +/− CT and Sgca −/− CT muscle. Scale bar =100 μm ( A and B ). C : 40 μg of SDS whole muscle lysate from gastrocnemius is loaded per lane. β, γ and δ sarcoglycan protein levels were not increased in Sgca −/− CT muscle as they were in Sgca +/− CT muscle. Utrophin, α dystroglycan, and plectin 1, by contrast, were similarly increased in both Sgca −/− CT and Sgca +/− CT muscle. Actin is shown as a control for protein loading and transfer.
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    Image Search Results


    Immunostaining of quadriceps muscle samples for Patients 1 and 2. Complete absence of staining (K and M) for  γ -sarcoglycan antibody is demonstrated in both patients. Patient 1′s muscle tissue (E, H, and N) shows partial reduction in staining of  α -,  β -, and  δ -sarcoglycan, while tissue on Patient 2 (F, I, and O) demonstrates loss of expression of  α - and  β -sarcoglycan and reduction in  δ -sarcoglycan.

    Journal: Molecular Genetics & Genomic Medicine

    Article Title: A slowly progressive form of limb-girdle muscular dystrophy type 2C associated with founder mutation in the SGCG gene in Puerto Rican Hispanics

    doi: 10.1002/mgg3.125

    Figure Lengend Snippet: Immunostaining of quadriceps muscle samples for Patients 1 and 2. Complete absence of staining (K and M) for γ -sarcoglycan antibody is demonstrated in both patients. Patient 1′s muscle tissue (E, H, and N) shows partial reduction in staining of α -, β -, and δ -sarcoglycan, while tissue on Patient 2 (F, I, and O) demonstrates loss of expression of α - and β -sarcoglycan and reduction in δ -sarcoglycan.

    Article Snippet: Immunofluorescence stains were done using the following antibodies: carboxyl terminus of dystrophin (Leica: NCL-DYS2 for Patient 1 and Abcam: ab15277 for Patient 2), and the sarcoglycans α - (Developmental Studies Hybridoma Bank, The University of Iowa), β -, γ -, and δ -sarcoglycan (Leica Biosystems, Newcastle Upon Tyne, UK).

    Techniques: Immunostaining, Staining, Expressing

    Isolation of alpha-sarcoglycan + EVs from plasma using immunoaffinity capturing. Anti-alpha-sarcoglycan antibodies were conjugated to magnetic beads to isolate muscle EVs from plasma. Western blot analysis confirmed the presence of the exosomal marker Tsg101 in isolated EVs. Ponceau S Staining has been used as loading control (a). MicroRNA quantifications showed an increase in the miR-206/miR-16 ratio in the SGCA + sub-population of EVs (SGCA-Beads) compared to total (total plasma EVs) or uncaptured EVs (Supernatant). MiR-206 expression levels were normalized versus the endogenous reference miR-16 and expressed as -ΔCq (where ΔCq = Cq miR-206 -Cq miR-16 ) (b). Moreover, the quantification of miR-16 ratio in the SGCA + sub-population of EVs compared to total or uncaptured EVs shows that SGCA-conjugated beads retained about 2–5% of the total amount of EVs, miR-16 expression levels were normalized versus the spike-in reference cel-miR-39 and expressed as -ΔCq (where ΔCq = Cq miR-16 -Cq cel-miR-39 ) (c). Asterisks denote significant changes ( p

    Journal: PLoS ONE

    Article Title: Muscle Releases Alpha-Sarcoglycan Positive Extracellular Vesicles Carrying miRNAs in the Bloodstream

    doi: 10.1371/journal.pone.0125094

    Figure Lengend Snippet: Isolation of alpha-sarcoglycan + EVs from plasma using immunoaffinity capturing. Anti-alpha-sarcoglycan antibodies were conjugated to magnetic beads to isolate muscle EVs from plasma. Western blot analysis confirmed the presence of the exosomal marker Tsg101 in isolated EVs. Ponceau S Staining has been used as loading control (a). MicroRNA quantifications showed an increase in the miR-206/miR-16 ratio in the SGCA + sub-population of EVs (SGCA-Beads) compared to total (total plasma EVs) or uncaptured EVs (Supernatant). MiR-206 expression levels were normalized versus the endogenous reference miR-16 and expressed as -ΔCq (where ΔCq = Cq miR-206 -Cq miR-16 ) (b). Moreover, the quantification of miR-16 ratio in the SGCA + sub-population of EVs compared to total or uncaptured EVs shows that SGCA-conjugated beads retained about 2–5% of the total amount of EVs, miR-16 expression levels were normalized versus the spike-in reference cel-miR-39 and expressed as -ΔCq (where ΔCq = Cq miR-16 -Cq cel-miR-39 ) (c). Asterisks denote significant changes ( p

    Article Snippet: The EVs were resuspended in PBS + 0.1% BSA and then stained with an anti-CD81 PE (clone BD Pharmingen, clone JS-81) or anti-alpha-sarcoglycan (clone AD1/20A6 Monosan) and labelled by goat anti-mouse (GaM) FITC.

    Techniques: Isolation, Magnetic Beads, Western Blot, Marker, Staining, Expressing

    Isolation of alpha-sarcoglycan + EVs from plasma using immunoaffinity capturing. Anti-alpha-sarcoglycan antibodies were conjugated to magnetic beads to isolate muscle EVs from plasma. Western blot analysis confirmed the presence of the exosomal marker Tsg101 in isolated EVs. Ponceau S Staining has been used as loading control (a). MicroRNA quantifications showed an increase in the miR-206/miR-16 ratio in the SGCA + sub-population of EVs (SGCA-Beads) compared to total (total plasma EVs) or uncaptured EVs (Supernatant). MiR-206 expression levels were normalized versus the endogenous reference miR-16 and expressed as -ΔCq (where ΔCq = Cq miR-206 -Cq miR-16 ) (b). Moreover, the quantification of miR-16 ratio in the SGCA + sub-population of EVs compared to total or uncaptured EVs shows that SGCA-conjugated beads retained about 2–5% of the total amount of EVs, miR-16 expression levels were normalized versus the spike-in reference cel-miR-39 and expressed as -ΔCq (where ΔCq = Cq miR-16 -Cq cel-miR-39 ) (c). Asterisks denote significant changes ( p

    Journal: PLoS ONE

    Article Title: Muscle Releases Alpha-Sarcoglycan Positive Extracellular Vesicles Carrying miRNAs in the Bloodstream

    doi: 10.1371/journal.pone.0125094

    Figure Lengend Snippet: Isolation of alpha-sarcoglycan + EVs from plasma using immunoaffinity capturing. Anti-alpha-sarcoglycan antibodies were conjugated to magnetic beads to isolate muscle EVs from plasma. Western blot analysis confirmed the presence of the exosomal marker Tsg101 in isolated EVs. Ponceau S Staining has been used as loading control (a). MicroRNA quantifications showed an increase in the miR-206/miR-16 ratio in the SGCA + sub-population of EVs (SGCA-Beads) compared to total (total plasma EVs) or uncaptured EVs (Supernatant). MiR-206 expression levels were normalized versus the endogenous reference miR-16 and expressed as -ΔCq (where ΔCq = Cq miR-206 -Cq miR-16 ) (b). Moreover, the quantification of miR-16 ratio in the SGCA + sub-population of EVs compared to total or uncaptured EVs shows that SGCA-conjugated beads retained about 2–5% of the total amount of EVs, miR-16 expression levels were normalized versus the spike-in reference cel-miR-39 and expressed as -ΔCq (where ΔCq = Cq miR-16 -Cq cel-miR-39 ) (c). Asterisks denote significant changes ( p

    Article Snippet: Primary antibodies were used against Tsg101 (1:2,000 dilution, clone 4A10 Abcam) and alpha-sarcoglycan (1:300 dilution, clone H-82 Santa Cruz).

    Techniques: Isolation, Magnetic Beads, Western Blot, Marker, Staining, Expressing

    Ameliorated NMJ fragmentation and denervation in aged mice by SGα overexpression. A , Reduced fragmentation and improved innervation. Shown were TA muscles of mice at different ages and indicated AAV treatment, whole-mount stained with CF568 α-BTX (red) and anti-NF/Syn antibodies (visualized by AlexaFluor 488 goat anti-rabbit IgG; green). Blue arrowhead, denervated or partially innervated endplate. Scale bar, 50 μm. B , Enlarged images of individual endplates. Scale bar, 10 μm. C – F , Quantitative analysis of data in A . C , Rescue of denervation in aged mice by SGα expression; F (2,12) = 29, *** p = 0.0001, ** p = 0.0051. D , Rescue of fragmentation number in aged mice by SGα expression; F (2,12) = 25.8, *** p = 0.0001, ** p = 0.0023. E , Rescue of fragmented endplate percentage in aged mice by SGα expression; F (2,12) = 93, *** p = 0.0001, *** p = 0.0003. F , Rescue of reduced AChR cluster intensity in aged mice by SGα expression; F (2,12) = 35.9, *** p = 0.0001, ** p = 0.0028; N = 5 mice per group, one-way ANOVA.

    Journal: The Journal of Neuroscience

    Article Title: Sarcoglycan Alpha Mitigates Neuromuscular Junction Decline in Aged Mice by Stabilizing LRP4

    doi: 10.1523/JNEUROSCI.0860-18.2018

    Figure Lengend Snippet: Ameliorated NMJ fragmentation and denervation in aged mice by SGα overexpression. A , Reduced fragmentation and improved innervation. Shown were TA muscles of mice at different ages and indicated AAV treatment, whole-mount stained with CF568 α-BTX (red) and anti-NF/Syn antibodies (visualized by AlexaFluor 488 goat anti-rabbit IgG; green). Blue arrowhead, denervated or partially innervated endplate. Scale bar, 50 μm. B , Enlarged images of individual endplates. Scale bar, 10 μm. C – F , Quantitative analysis of data in A . C , Rescue of denervation in aged mice by SGα expression; F (2,12) = 29, *** p = 0.0001, ** p = 0.0051. D , Rescue of fragmentation number in aged mice by SGα expression; F (2,12) = 25.8, *** p = 0.0001, ** p = 0.0023. E , Rescue of fragmented endplate percentage in aged mice by SGα expression; F (2,12) = 93, *** p = 0.0001, *** p = 0.0003. F , Rescue of reduced AChR cluster intensity in aged mice by SGα expression; F (2,12) = 35.9, *** p = 0.0001, ** p = 0.0028; N = 5 mice per group, one-way ANOVA.

    Article Snippet: Antibodies used were as follows: AChRδ (88B; 1:2000 for Western blot) from ThermoFisher Scientific; AChRε (ab65180; 1:2000 for Western blot), SGα (ab189254; 1:500 for staining and 1:2000 for Western blot) from Abcam; Ubiquitin (sc-8017; 1:1000 for Western blot), AChRα (sc-65829; 1:1000 for Western blot), and AChRβ (sc-11371; 1:1000 for Western blot) from Santa Cruz Biotechnology; DOK7 (AF6398; 1:1000 for Western blot) from R & D Systems; neurofilament (C28E10; 1:500 for staining) and synapsin (D12G5, 1:500 for staining) from Cell Signaling Technology; GAPDH (NB 600-501; 1:3000 for Western blot) from Novus; V5 (V8012; 1:1000 for Western blot), laminin (041M4799; 1:200 for staining) and GFP (11814460001; 1:3000 for Western blot and 1:500 for staining) from Sigma Aldrich.

    Techniques: Mouse Assay, Over Expression, Staining, Expressing

    Restored muscle size and strength in aged mice by SGα expression. A , Increased cross-section area and reduced central nuclei in aged muscle by SGα expression. Cross-sections of TA muscle were stained with laminin antibody (red) and DAPI (blue). Yellow arrowhead, central nuclei. Scale bar, 20 μm. B , Increased cross-section area in aged mice by SGα expression; F (2,9) = 24.1, *** p = 0.0003, ** p = 0.0012. C , Reduced central nuclei in aged mice by SGα expression; F (2,9) = 27.8, *** p = 0.0002, *** p = 0.0006; N = 4 mice per group, one-way ANOVA. D , Representative twitch (top) and tetanic force at 50 and 150 Hz (bottom) by sciatic nerve stimulation. E , Rescue of reduced twitch force in aged mice by SGα expression; F (2,9) = 35.1, *** p = 0.0001 and 0.0008. F , Rescue of reduced tetanic force in aged mice by SGα expression; F (2,36) = 34, * p

    Journal: The Journal of Neuroscience

    Article Title: Sarcoglycan Alpha Mitigates Neuromuscular Junction Decline in Aged Mice by Stabilizing LRP4

    doi: 10.1523/JNEUROSCI.0860-18.2018

    Figure Lengend Snippet: Restored muscle size and strength in aged mice by SGα expression. A , Increased cross-section area and reduced central nuclei in aged muscle by SGα expression. Cross-sections of TA muscle were stained with laminin antibody (red) and DAPI (blue). Yellow arrowhead, central nuclei. Scale bar, 20 μm. B , Increased cross-section area in aged mice by SGα expression; F (2,9) = 24.1, *** p = 0.0003, ** p = 0.0012. C , Reduced central nuclei in aged mice by SGα expression; F (2,9) = 27.8, *** p = 0.0002, *** p = 0.0006; N = 4 mice per group, one-way ANOVA. D , Representative twitch (top) and tetanic force at 50 and 150 Hz (bottom) by sciatic nerve stimulation. E , Rescue of reduced twitch force in aged mice by SGα expression; F (2,9) = 35.1, *** p = 0.0001 and 0.0008. F , Rescue of reduced tetanic force in aged mice by SGα expression; F (2,36) = 34, * p

    Article Snippet: Antibodies used were as follows: AChRδ (88B; 1:2000 for Western blot) from ThermoFisher Scientific; AChRε (ab65180; 1:2000 for Western blot), SGα (ab189254; 1:500 for staining and 1:2000 for Western blot) from Abcam; Ubiquitin (sc-8017; 1:1000 for Western blot), AChRα (sc-65829; 1:1000 for Western blot), and AChRβ (sc-11371; 1:1000 for Western blot) from Santa Cruz Biotechnology; DOK7 (AF6398; 1:1000 for Western blot) from R & D Systems; neurofilament (C28E10; 1:500 for staining) and synapsin (D12G5, 1:500 for staining) from Cell Signaling Technology; GAPDH (NB 600-501; 1:3000 for Western blot) from Novus; V5 (V8012; 1:1000 for Western blot), laminin (041M4799; 1:200 for staining) and GFP (11814460001; 1:3000 for Western blot and 1:500 for staining) from Sigma Aldrich.

    Techniques: Mouse Assay, Expressing, Staining

    Increasing LRP4 protein stability by SGα. A , LRP4 interaction with SGα, but not SGδ. Flag-LRP4 was precipitated from HEK293 cells cotransfected with SGα-V5 or SGδ-V5 and probed with anti-V5 antibody. Lysates were probed with the same antibodies as control. B , In vivo interaction between LRP4 and SGα. Homogenates were prepared from muscles of Flag-Lrp4 transgenic mice and subjected to IP with anti-SGα antibody. Immunocomplex was probed with anti-Flag and SGα antibodies. C , mRNA expression of sarcoglycan complex molecules in diaphragm synaptic region; t (4) = 9.97, ** p = 0.0026 for Sgca ; t (4) = 8.87, ** p = 0.0039 for Sgcb ; t (4) = 10.8, *** p = 0.0004 for Sgcg ; N = 3 mice per group, unpaired t test. D , Reduced SGα protein level in aged mice. Top, Representative blots. Bottom, Quantitative data; t (4) = 5.66, * p = 0.015; N = 3 mice per group, unpaired t test. E , Discontinuous staining of SGα in aged TA muscles. White arrowhead, NMJs identified by α-BTX with discontinuous SGα staining. Scale bar, 20 μm. F , G , Increased LRP4 stability in HEK293 cells expressing SGα. HEK293 cells were transfected with Flag-LRP4 without (control) or with SGα-V5 and incubated with CHX. Flag-LRP4 levels were analyzed at indicated times. F , Representative blot; G , quantitative data; F (1,16) = 56.5, ** p = 0.0021 and 0.0095, *** p = 0.0001; N = 3, two-way ANOVA.

    Journal: The Journal of Neuroscience

    Article Title: Sarcoglycan Alpha Mitigates Neuromuscular Junction Decline in Aged Mice by Stabilizing LRP4

    doi: 10.1523/JNEUROSCI.0860-18.2018

    Figure Lengend Snippet: Increasing LRP4 protein stability by SGα. A , LRP4 interaction with SGα, but not SGδ. Flag-LRP4 was precipitated from HEK293 cells cotransfected with SGα-V5 or SGδ-V5 and probed with anti-V5 antibody. Lysates were probed with the same antibodies as control. B , In vivo interaction between LRP4 and SGα. Homogenates were prepared from muscles of Flag-Lrp4 transgenic mice and subjected to IP with anti-SGα antibody. Immunocomplex was probed with anti-Flag and SGα antibodies. C , mRNA expression of sarcoglycan complex molecules in diaphragm synaptic region; t (4) = 9.97, ** p = 0.0026 for Sgca ; t (4) = 8.87, ** p = 0.0039 for Sgcb ; t (4) = 10.8, *** p = 0.0004 for Sgcg ; N = 3 mice per group, unpaired t test. D , Reduced SGα protein level in aged mice. Top, Representative blots. Bottom, Quantitative data; t (4) = 5.66, * p = 0.015; N = 3 mice per group, unpaired t test. E , Discontinuous staining of SGα in aged TA muscles. White arrowhead, NMJs identified by α-BTX with discontinuous SGα staining. Scale bar, 20 μm. F , G , Increased LRP4 stability in HEK293 cells expressing SGα. HEK293 cells were transfected with Flag-LRP4 without (control) or with SGα-V5 and incubated with CHX. Flag-LRP4 levels were analyzed at indicated times. F , Representative blot; G , quantitative data; F (1,16) = 56.5, ** p = 0.0021 and 0.0095, *** p = 0.0001; N = 3, two-way ANOVA.

    Article Snippet: Antibodies used were as follows: AChRδ (88B; 1:2000 for Western blot) from ThermoFisher Scientific; AChRε (ab65180; 1:2000 for Western blot), SGα (ab189254; 1:500 for staining and 1:2000 for Western blot) from Abcam; Ubiquitin (sc-8017; 1:1000 for Western blot), AChRα (sc-65829; 1:1000 for Western blot), and AChRβ (sc-11371; 1:1000 for Western blot) from Santa Cruz Biotechnology; DOK7 (AF6398; 1:1000 for Western blot) from R & D Systems; neurofilament (C28E10; 1:500 for staining) and synapsin (D12G5, 1:500 for staining) from Cell Signaling Technology; GAPDH (NB 600-501; 1:3000 for Western blot) from Novus; V5 (V8012; 1:1000 for Western blot), laminin (041M4799; 1:200 for staining) and GFP (11814460001; 1:3000 for Western blot and 1:500 for staining) from Sigma Aldrich.

    Techniques: In Vivo, Transgenic Assay, Mouse Assay, Expressing, Staining, Transfection, Incubation

    Increased LRP4 level with reduced ubiquitination in aged muscles by SGα expression. A , Diagram of AAV9-SGα-GFP construct. Human SGCA was inserted at N-terminus of GFP as a fusion protein. ITR, Inverted terminal repeats; CMV, cytomegalovirus promoter; β-Glob, β-globulin intron. B , SGα expression in HEK293 cells transfected with AAV9-SGα-GFP. C , Expression of GFP in muscles after intramuscular and intravenous injection with indicated AAV. Shown were TA cross-sections. D , High infection rates by AAV9-expressing SGα-GFP. E , Increased protein level and reduced ubiquitination of LRP4 by SGα expression. Muscles were isolated 6 weeks after intravenous injection of AAV9-expressing SGα. F , G , Quantification of data in E ; F (2,6) = 16.7, ** p = 0.0029, * p = 0.035 for F ; F (2,6) = 29.7, ** p = 0.0021, *** p = 0.0009 for G ; N = 3 mice per group, one-way ANOVA.

    Journal: The Journal of Neuroscience

    Article Title: Sarcoglycan Alpha Mitigates Neuromuscular Junction Decline in Aged Mice by Stabilizing LRP4

    doi: 10.1523/JNEUROSCI.0860-18.2018

    Figure Lengend Snippet: Increased LRP4 level with reduced ubiquitination in aged muscles by SGα expression. A , Diagram of AAV9-SGα-GFP construct. Human SGCA was inserted at N-terminus of GFP as a fusion protein. ITR, Inverted terminal repeats; CMV, cytomegalovirus promoter; β-Glob, β-globulin intron. B , SGα expression in HEK293 cells transfected with AAV9-SGα-GFP. C , Expression of GFP in muscles after intramuscular and intravenous injection with indicated AAV. Shown were TA cross-sections. D , High infection rates by AAV9-expressing SGα-GFP. E , Increased protein level and reduced ubiquitination of LRP4 by SGα expression. Muscles were isolated 6 weeks after intravenous injection of AAV9-expressing SGα. F , G , Quantification of data in E ; F (2,6) = 16.7, ** p = 0.0029, * p = 0.035 for F ; F (2,6) = 29.7, ** p = 0.0021, *** p = 0.0009 for G ; N = 3 mice per group, one-way ANOVA.

    Article Snippet: Antibodies used were as follows: AChRδ (88B; 1:2000 for Western blot) from ThermoFisher Scientific; AChRε (ab65180; 1:2000 for Western blot), SGα (ab189254; 1:500 for staining and 1:2000 for Western blot) from Abcam; Ubiquitin (sc-8017; 1:1000 for Western blot), AChRα (sc-65829; 1:1000 for Western blot), and AChRβ (sc-11371; 1:1000 for Western blot) from Santa Cruz Biotechnology; DOK7 (AF6398; 1:1000 for Western blot) from R & D Systems; neurofilament (C28E10; 1:500 for staining) and synapsin (D12G5, 1:500 for staining) from Cell Signaling Technology; GAPDH (NB 600-501; 1:3000 for Western blot) from Novus; V5 (V8012; 1:1000 for Western blot), laminin (041M4799; 1:200 for staining) and GFP (11814460001; 1:3000 for Western blot and 1:500 for staining) from Sigma Aldrich.

    Techniques: Expressing, Construct, Transfection, Injection, Infection, Isolation, Mouse Assay

    Improved neuromuscular transmission in aged mice by SGα expression. A , B , Reduced ratio of 2nd–10th CMAP amplitudes over the first CMAP amplitude at 40 Hz stimulations. A , Representative traces; B , quantitative data; F (2,90) = 47.7, * p

    Journal: The Journal of Neuroscience

    Article Title: Sarcoglycan Alpha Mitigates Neuromuscular Junction Decline in Aged Mice by Stabilizing LRP4

    doi: 10.1523/JNEUROSCI.0860-18.2018

    Figure Lengend Snippet: Improved neuromuscular transmission in aged mice by SGα expression. A , B , Reduced ratio of 2nd–10th CMAP amplitudes over the first CMAP amplitude at 40 Hz stimulations. A , Representative traces; B , quantitative data; F (2,90) = 47.7, * p

    Article Snippet: Antibodies used were as follows: AChRδ (88B; 1:2000 for Western blot) from ThermoFisher Scientific; AChRε (ab65180; 1:2000 for Western blot), SGα (ab189254; 1:500 for staining and 1:2000 for Western blot) from Abcam; Ubiquitin (sc-8017; 1:1000 for Western blot), AChRα (sc-65829; 1:1000 for Western blot), and AChRβ (sc-11371; 1:1000 for Western blot) from Santa Cruz Biotechnology; DOK7 (AF6398; 1:1000 for Western blot) from R & D Systems; neurofilament (C28E10; 1:500 for staining) and synapsin (D12G5, 1:500 for staining) from Cell Signaling Technology; GAPDH (NB 600-501; 1:3000 for Western blot) from Novus; V5 (V8012; 1:1000 for Western blot), laminin (041M4799; 1:200 for staining) and GFP (11814460001; 1:3000 for Western blot and 1:500 for staining) from Sigma Aldrich.

    Techniques: Transmission Assay, Mouse Assay, Expressing

    Corrector C17 induced a dose dependent increase of α-SG mutant in LGMD2D myotubes. ( A ) representative western blot of total protein lysates of myogenic cells from a patient carrying the L31P/V247M α-SG mutations grown and differentiated for 7 days and treated for the last 48 h with either 1‰ DMSO (vehicle) or increasing concentrations of corrector C17, as indicated. α-SG protein was revealed with specific primary antibody, the Ponceau red staining (PR) is reported and utilized to normalize the total amount of proteins loaded in each lane. ( B ) quantification by densitometric analysis of α-SG protein bands of three independent western blot experiments, as described in (A). The average amount of α-SG (± SEM) is expressed as fold increase of the protein content present in myotubes treated with vehicle. Statistical analysis was performed by One-way ANOVA test - multiple comparisons Dunnett test; * P ≤ 0.05; ** P ≤ 0.01. ( C ) myogenic cells from a healthy subject were grown and differentiated for 7 days and treated for the last 48 hours with either 1‰ DMSO (vehicle) or 15 µM C17. Total protein lysates were analyzed by Western blot as described in (A). ( D ) quantification by densitometric analysis of wild type α-SG protein bands of three independent Western blot experiments as described in (C). Statistical analysis was performed by unpaired two-tailed Student’s t -test.

    Journal: Human Molecular Genetics

    Article Title: Repairing folding-defective α-sarcoglycan mutants by CFTR correctors, a potential therapy for limb-girdle muscular dystrophy 2D

    doi: 10.1093/hmg/ddy013

    Figure Lengend Snippet: Corrector C17 induced a dose dependent increase of α-SG mutant in LGMD2D myotubes. ( A ) representative western blot of total protein lysates of myogenic cells from a patient carrying the L31P/V247M α-SG mutations grown and differentiated for 7 days and treated for the last 48 h with either 1‰ DMSO (vehicle) or increasing concentrations of corrector C17, as indicated. α-SG protein was revealed with specific primary antibody, the Ponceau red staining (PR) is reported and utilized to normalize the total amount of proteins loaded in each lane. ( B ) quantification by densitometric analysis of α-SG protein bands of three independent western blot experiments, as described in (A). The average amount of α-SG (± SEM) is expressed as fold increase of the protein content present in myotubes treated with vehicle. Statistical analysis was performed by One-way ANOVA test - multiple comparisons Dunnett test; * P ≤ 0.05; ** P ≤ 0.01. ( C ) myogenic cells from a healthy subject were grown and differentiated for 7 days and treated for the last 48 hours with either 1‰ DMSO (vehicle) or 15 µM C17. Total protein lysates were analyzed by Western blot as described in (A). ( D ) quantification by densitometric analysis of wild type α-SG protein bands of three independent Western blot experiments as described in (C). Statistical analysis was performed by unpaired two-tailed Student’s t -test.

    Article Snippet: Antibodies Mouse monoclonal antibody specific for α-SG (NCL-a-SARC) was from Leica Biosystem; rabbit monoclonal anti α-SG (AB189254) was from Abcam; mouse monoclonal antibody specific for β-SG, δ-SG, γ-SG and β-actin were from Sigma, rabbit polyclonal antibody specific for α-and δ-SG were produced as previously described , rabbit polyclonal antibody specific for α-actinin was from Santa Cruz.

    Techniques: Mutagenesis, Western Blot, Staining, Two Tailed Test

    Corrector C17 had no effect on the transcription of SGCA and stabilized α-SG mutant, in LGMD2D myotubes. ( A ) LGMD2D myotubes were treated for 48h with vehicle (1‰ DMSO) or 15 µM C17. The SGCA transcription was evaluated by quantitative real-time PCR (see M M for details concerning normalization) and reported as mean, ± SEM, relative to DMSO treated control of two independent experiments performed in quadruplicate. Statistical analysis was performed using unpaired two-tailed Student’s t -test; n.s., P > 0.05. ( B ) myogenic cells from a patient carrying the L31P/V247M α-SG mutations were grown and differentiated for 7 days and treated with 1‰ DMSO (vehicle) or 15 µM C17 for 96 h. At the end of incubation, 100 µg/ml cycloheximide was added and myotubes were lysate at the indicated time points. Protein lysate were analyzed by western blot with anti α-SG antibody, the Ponceau red staining (PR) is reported to normalize the total amount of proteins loaded in each lane. ( C ) quantification by densitometric analysis of α-SG protein bands of three independent western blot experiments, as described in (B). The average amount of α-SG (± SEM) is expressed as percentage of the protein present at time 0. Statistical analysis was performed by unpaired two-tailed Student’s t -test; * P ≤ 0.05; ** P ≤ 0.01.

    Journal: Human Molecular Genetics

    Article Title: Repairing folding-defective α-sarcoglycan mutants by CFTR correctors, a potential therapy for limb-girdle muscular dystrophy 2D

    doi: 10.1093/hmg/ddy013

    Figure Lengend Snippet: Corrector C17 had no effect on the transcription of SGCA and stabilized α-SG mutant, in LGMD2D myotubes. ( A ) LGMD2D myotubes were treated for 48h with vehicle (1‰ DMSO) or 15 µM C17. The SGCA transcription was evaluated by quantitative real-time PCR (see M M for details concerning normalization) and reported as mean, ± SEM, relative to DMSO treated control of two independent experiments performed in quadruplicate. Statistical analysis was performed using unpaired two-tailed Student’s t -test; n.s., P > 0.05. ( B ) myogenic cells from a patient carrying the L31P/V247M α-SG mutations were grown and differentiated for 7 days and treated with 1‰ DMSO (vehicle) or 15 µM C17 for 96 h. At the end of incubation, 100 µg/ml cycloheximide was added and myotubes were lysate at the indicated time points. Protein lysate were analyzed by western blot with anti α-SG antibody, the Ponceau red staining (PR) is reported to normalize the total amount of proteins loaded in each lane. ( C ) quantification by densitometric analysis of α-SG protein bands of three independent western blot experiments, as described in (B). The average amount of α-SG (± SEM) is expressed as percentage of the protein present at time 0. Statistical analysis was performed by unpaired two-tailed Student’s t -test; * P ≤ 0.05; ** P ≤ 0.01.

    Article Snippet: Antibodies Mouse monoclonal antibody specific for α-SG (NCL-a-SARC) was from Leica Biosystem; rabbit monoclonal anti α-SG (AB189254) was from Abcam; mouse monoclonal antibody specific for β-SG, δ-SG, γ-SG and β-actin were from Sigma, rabbit polyclonal antibody specific for α-and δ-SG were produced as previously described , rabbit polyclonal antibody specific for α-actinin was from Santa Cruz.

    Techniques: Mutagenesis, Real-time Polymerase Chain Reaction, Two Tailed Test, Incubation, Western Blot, Staining

    Rescue of the folding-defective R77C-α-SG by means of CFTR correctors. ( A ) Western blot of protein lysates from βγδ-cells transiently expressing R77C-α-SG and treated for 24 h with corrector C5 5 µM, C9 10 µM, C17 10 µM, C4 5 µM. One sample was treated with the combination of corrector C17 and C4 (each one-half dose). Cells expressing wild type-α-SG were utilized as positive control. Membrane were probed with antibodies against α-SG and α-actinin, used as loading control. ( B ) quantification by densitometric analysis of α-SG protein bands on at least three independent Western blot experiments. The average amount of α-SG (± SEM) is shown as percentage of the protein content in cells expressing the wild type form. Statistical analysis was performed by One-way ANOVA test - multiple comparisons Dunnett test; ** P ≤ 0.01; **** P ≤ 0.0001. ( C ) IF confocal analysis of βγδ-cells expressing R77C-α-SG and treated for 24 h with the indicated correctors. Intact cells (not permeabilized) were immune-decorated with an anti α-SG antibody, recognizing an extracellular epitope, revealed by the secondary Alexa Fluor 594-conjugated anti-mouse antibody. Cells expressing wild type-α-SG are shown as positive control. On the right of each image is reported the same field with nuclei stained by DAPI. Images were recorded with a Leica SP5 laser scanning confocal microscope at the same setting conditions and magnification. ( D ) mean fluorescence intensity of membrane staining of βγδ-cells expressing R77C-α-SG treated for 24 h with vehicle (negative control) or the indicated correctors; βγδ-cells expressing WT-α-SG were used as positive control. Fluorescence values from at least three independent experiments, performed in triplicate, were recorded by using the ImageXpress microscope system. Mean values (± SEM) were normalized for the number of cells positive for both α-SG and DAPI under permeabilization condition to consider transfection efficiency. Statistical analysis was performed by One-way ANOVA test - multiple comparisons Bonferroni test; n.s., P > 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001.

    Journal: Human Molecular Genetics

    Article Title: Repairing folding-defective α-sarcoglycan mutants by CFTR correctors, a potential therapy for limb-girdle muscular dystrophy 2D

    doi: 10.1093/hmg/ddy013

    Figure Lengend Snippet: Rescue of the folding-defective R77C-α-SG by means of CFTR correctors. ( A ) Western blot of protein lysates from βγδ-cells transiently expressing R77C-α-SG and treated for 24 h with corrector C5 5 µM, C9 10 µM, C17 10 µM, C4 5 µM. One sample was treated with the combination of corrector C17 and C4 (each one-half dose). Cells expressing wild type-α-SG were utilized as positive control. Membrane were probed with antibodies against α-SG and α-actinin, used as loading control. ( B ) quantification by densitometric analysis of α-SG protein bands on at least three independent Western blot experiments. The average amount of α-SG (± SEM) is shown as percentage of the protein content in cells expressing the wild type form. Statistical analysis was performed by One-way ANOVA test - multiple comparisons Dunnett test; ** P ≤ 0.01; **** P ≤ 0.0001. ( C ) IF confocal analysis of βγδ-cells expressing R77C-α-SG and treated for 24 h with the indicated correctors. Intact cells (not permeabilized) were immune-decorated with an anti α-SG antibody, recognizing an extracellular epitope, revealed by the secondary Alexa Fluor 594-conjugated anti-mouse antibody. Cells expressing wild type-α-SG are shown as positive control. On the right of each image is reported the same field with nuclei stained by DAPI. Images were recorded with a Leica SP5 laser scanning confocal microscope at the same setting conditions and magnification. ( D ) mean fluorescence intensity of membrane staining of βγδ-cells expressing R77C-α-SG treated for 24 h with vehicle (negative control) or the indicated correctors; βγδ-cells expressing WT-α-SG were used as positive control. Fluorescence values from at least three independent experiments, performed in triplicate, were recorded by using the ImageXpress microscope system. Mean values (± SEM) were normalized for the number of cells positive for both α-SG and DAPI under permeabilization condition to consider transfection efficiency. Statistical analysis was performed by One-way ANOVA test - multiple comparisons Bonferroni test; n.s., P > 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001.

    Article Snippet: Antibodies Mouse monoclonal antibody specific for α-SG (NCL-a-SARC) was from Leica Biosystem; rabbit monoclonal anti α-SG (AB189254) was from Abcam; mouse monoclonal antibody specific for β-SG, δ-SG, γ-SG and β-actin were from Sigma, rabbit polyclonal antibody specific for α-and δ-SG were produced as previously described , rabbit polyclonal antibody specific for α-actinin was from Santa Cruz.

    Techniques: Western Blot, Expressing, Positive Control, Staining, Microscopy, Fluorescence, Negative Control, Transfection

    CFTR correctors promoted increase and traffic of R98H-α-sarcoglycan. ( A ) Representative western blot of total protein lysates from HEK-293 cells transiently expressing the R98H form of α-SG and treated with the indicated CFTR correctors, at the concentrations reported in Table 1 , MG132 10 µM, as in ( 6 ) or vehicle (1‰ DMSO); lysate from cells expressing wild type-α-SG was used for comparison. Membranes were incubated with primary antibodies against α-SG and β-actin, used as loading control; arrowhead indicates the de-glycosylated form of the protein ( 6 ), whereas asterisks indicate the immature form of the protein recognized by the α-SG antibody. ( B ) Quantification of α-SG content by densitometric analysis of western blots from at least four independent experiments. The average amount of α-SG (± SEM), is expressed as fold increase of the protein content compared with the negative control (vehicle). Statistical analysis was performed by One-way ANOVA test - multiple comparisons Dunnett test; n.s., P > 0.05; * P ≤ 0.05; ** P ≤ 0.01. ( C ) Membrane localization of α-SG in HEK293 cells expressing R98H-α-SG treated with either vehicle or the indicated CFTR correctors. For comparison, cells expressing wild type-α-SG were also analysed. Localization was evaluated by confocal immunofluorescence analysis of intact cells immuno-decorated with an antibody recognizing an extracellular epitope of α-SG. The primary antibody was revealed with the secondary Alexa Fluor 594-conjugated anti-mouse antibody. Images were recorded with a Leica SP5 laser scanning confocal microscope at the same setting conditions and magnification. Below each image the same field in light transmission was recorded.

    Journal: Human Molecular Genetics

    Article Title: Repairing folding-defective α-sarcoglycan mutants by CFTR correctors, a potential therapy for limb-girdle muscular dystrophy 2D

    doi: 10.1093/hmg/ddy013

    Figure Lengend Snippet: CFTR correctors promoted increase and traffic of R98H-α-sarcoglycan. ( A ) Representative western blot of total protein lysates from HEK-293 cells transiently expressing the R98H form of α-SG and treated with the indicated CFTR correctors, at the concentrations reported in Table 1 , MG132 10 µM, as in ( 6 ) or vehicle (1‰ DMSO); lysate from cells expressing wild type-α-SG was used for comparison. Membranes were incubated with primary antibodies against α-SG and β-actin, used as loading control; arrowhead indicates the de-glycosylated form of the protein ( 6 ), whereas asterisks indicate the immature form of the protein recognized by the α-SG antibody. ( B ) Quantification of α-SG content by densitometric analysis of western blots from at least four independent experiments. The average amount of α-SG (± SEM), is expressed as fold increase of the protein content compared with the negative control (vehicle). Statistical analysis was performed by One-way ANOVA test - multiple comparisons Dunnett test; n.s., P > 0.05; * P ≤ 0.05; ** P ≤ 0.01. ( C ) Membrane localization of α-SG in HEK293 cells expressing R98H-α-SG treated with either vehicle or the indicated CFTR correctors. For comparison, cells expressing wild type-α-SG were also analysed. Localization was evaluated by confocal immunofluorescence analysis of intact cells immuno-decorated with an antibody recognizing an extracellular epitope of α-SG. The primary antibody was revealed with the secondary Alexa Fluor 594-conjugated anti-mouse antibody. Images were recorded with a Leica SP5 laser scanning confocal microscope at the same setting conditions and magnification. Below each image the same field in light transmission was recorded.

    Article Snippet: Antibodies Mouse monoclonal antibody specific for α-SG (NCL-a-SARC) was from Leica Biosystem; rabbit monoclonal anti α-SG (AB189254) was from Abcam; mouse monoclonal antibody specific for β-SG, δ-SG, γ-SG and β-actin were from Sigma, rabbit polyclonal antibody specific for α-and δ-SG were produced as previously described , rabbit polyclonal antibody specific for α-actinin was from Santa Cruz.

    Techniques: Western Blot, Expressing, Incubation, Negative Control, Immunofluorescence, Microscopy, Transmission Assay

    C17 treatment restores membrane functionality to patient’s myotubes in vitro . Myogenic cells from a patient carrying the L31P/V247M α-SG mutations were grown and differentiated for 7 days and treated for the last 96 h with 1‰ DMSO (vehicle) or 15 µM C17. At the end of the treatment, myotubes were incubated for 20 min in hypo-osmotic solutions as indicated. Then, the cytosolic protein creatine kinase (CK) was measured in the supernatant of myotubes, whereas the intracellular level of the protein was determined after cell lysis. Ratios between extra and total CK values were plotted as average ± SEM of two independent experiments performed in sextuplicate. As reference, the release of CK from myotubes from a healthy subject was assessed at the same hypo-osmotic conditions. Statistical analysis was performed using One-way ANOVA test—multiple comparisons Dunnett test; ** P ≤ 0.01; *** P ≤ 0.001.

    Journal: Human Molecular Genetics

    Article Title: Repairing folding-defective α-sarcoglycan mutants by CFTR correctors, a potential therapy for limb-girdle muscular dystrophy 2D

    doi: 10.1093/hmg/ddy013

    Figure Lengend Snippet: C17 treatment restores membrane functionality to patient’s myotubes in vitro . Myogenic cells from a patient carrying the L31P/V247M α-SG mutations were grown and differentiated for 7 days and treated for the last 96 h with 1‰ DMSO (vehicle) or 15 µM C17. At the end of the treatment, myotubes were incubated for 20 min in hypo-osmotic solutions as indicated. Then, the cytosolic protein creatine kinase (CK) was measured in the supernatant of myotubes, whereas the intracellular level of the protein was determined after cell lysis. Ratios between extra and total CK values were plotted as average ± SEM of two independent experiments performed in sextuplicate. As reference, the release of CK from myotubes from a healthy subject was assessed at the same hypo-osmotic conditions. Statistical analysis was performed using One-way ANOVA test—multiple comparisons Dunnett test; ** P ≤ 0.01; *** P ≤ 0.001.

    Article Snippet: Antibodies Mouse monoclonal antibody specific for α-SG (NCL-a-SARC) was from Leica Biosystem; rabbit monoclonal anti α-SG (AB189254) was from Abcam; mouse monoclonal antibody specific for β-SG, δ-SG, γ-SG and β-actin were from Sigma, rabbit polyclonal antibody specific for α-and δ-SG were produced as previously described , rabbit polyclonal antibody specific for α-actinin was from Santa Cruz.

    Techniques: In Vitro, Incubation, Lysis

    CFTR correctors rescued the sarcoglycan complex in LGMD2D myotubes. Myogenic cells from a patient carrying the L31P/V247M α-SG mutations were grown and differentiated for 7 days and treated for the last 48 h with 1‰ DMSO (vehicle) or the indicated CFTR correctors. At the end of incubation intact myotubes (not permeabilized) were labelled with antibodies recognizing an extracellular epitope of either α-SG (on the left) or δ-SG (on the right), as indicated, to mark the membrane resident sarcoglycans only. Primary antibodies were revealed with the secondary DyLight 488-conjugated anti-rabbit antibodies. Bars indicate 31.75 µm. Images were recorded with a Leica SP5 laser scanning confocal microscope at the same setting conditions.

    Journal: Human Molecular Genetics

    Article Title: Repairing folding-defective α-sarcoglycan mutants by CFTR correctors, a potential therapy for limb-girdle muscular dystrophy 2D

    doi: 10.1093/hmg/ddy013

    Figure Lengend Snippet: CFTR correctors rescued the sarcoglycan complex in LGMD2D myotubes. Myogenic cells from a patient carrying the L31P/V247M α-SG mutations were grown and differentiated for 7 days and treated for the last 48 h with 1‰ DMSO (vehicle) or the indicated CFTR correctors. At the end of incubation intact myotubes (not permeabilized) were labelled with antibodies recognizing an extracellular epitope of either α-SG (on the left) or δ-SG (on the right), as indicated, to mark the membrane resident sarcoglycans only. Primary antibodies were revealed with the secondary DyLight 488-conjugated anti-rabbit antibodies. Bars indicate 31.75 µm. Images were recorded with a Leica SP5 laser scanning confocal microscope at the same setting conditions.

    Article Snippet: Antibodies Mouse monoclonal antibody specific for α-SG (NCL-a-SARC) was from Leica Biosystem; rabbit monoclonal anti α-SG (AB189254) was from Abcam; mouse monoclonal antibody specific for β-SG, δ-SG, γ-SG and β-actin were from Sigma, rabbit polyclonal antibody specific for α-and δ-SG were produced as previously described , rabbit polyclonal antibody specific for α-actinin was from Santa Cruz.

    Techniques: Incubation, Microscopy

    Corrector C17 induced a time dependent increase of mutated α-SG, without toxicity, in LGMD2D myotubes. ( A ) myogenic cells from a patient carrying the L31P/V247M α-SG mutations were grown and differentiated for 7 days and treated with 1‰ DMSO (vehicle) or 15 µM C17 for the indicated time intervals. α-SG protein content was evaluated by western blot of total myotube lysates. The Ponceau red staining (PR) is reported to normalize the total amount of proteins loaded in each lane. ( B ) quantification by densitometric analysis of α-SG protein bands of three independent western blot experiments, as described in (A). The average amount of α-SG (± SEM) is expressed as fold increase of the protein content present in myotubes treated with vehicle for the same incubation interval. Statistical analysis was performed by One-way ANOVA test - multiple comparisons Bonferroni test; * P ≤ 0.05; *** P ≤ 0.001. ( C ) phase contrast images of myotubes treated with either 1‰ DMSO (vehicle) or C17 at the concentration and time intervals indicated to evaluate possible toxic effects. All images were recorded at the same magnification.

    Journal: Human Molecular Genetics

    Article Title: Repairing folding-defective α-sarcoglycan mutants by CFTR correctors, a potential therapy for limb-girdle muscular dystrophy 2D

    doi: 10.1093/hmg/ddy013

    Figure Lengend Snippet: Corrector C17 induced a time dependent increase of mutated α-SG, without toxicity, in LGMD2D myotubes. ( A ) myogenic cells from a patient carrying the L31P/V247M α-SG mutations were grown and differentiated for 7 days and treated with 1‰ DMSO (vehicle) or 15 µM C17 for the indicated time intervals. α-SG protein content was evaluated by western blot of total myotube lysates. The Ponceau red staining (PR) is reported to normalize the total amount of proteins loaded in each lane. ( B ) quantification by densitometric analysis of α-SG protein bands of three independent western blot experiments, as described in (A). The average amount of α-SG (± SEM) is expressed as fold increase of the protein content present in myotubes treated with vehicle for the same incubation interval. Statistical analysis was performed by One-way ANOVA test - multiple comparisons Bonferroni test; * P ≤ 0.05; *** P ≤ 0.001. ( C ) phase contrast images of myotubes treated with either 1‰ DMSO (vehicle) or C17 at the concentration and time intervals indicated to evaluate possible toxic effects. All images were recorded at the same magnification.

    Article Snippet: Antibodies Mouse monoclonal antibody specific for α-SG (NCL-a-SARC) was from Leica Biosystem; rabbit monoclonal anti α-SG (AB189254) was from Abcam; mouse monoclonal antibody specific for β-SG, δ-SG, γ-SG and β-actin were from Sigma, rabbit polyclonal antibody specific for α-and δ-SG were produced as previously described , rabbit polyclonal antibody specific for α-actinin was from Santa Cruz.

    Techniques: Western Blot, Staining, Incubation, Concentration Assay

    Corrector C6, C5 and C17 induced a dose-dependent increase of different α-SG mutants without major toxic effects. ( A , D , G ) Quantification of α-SG content in HEK293 cells expressing R98H-α-SG (A), D97G-α-SG (D) or V247M-α-SG (G) treated for 24 h with increasing concentrations of the indicated correctors. α-SG protein content was determined by WB and densitometric analysis on total protein lysates from at least three independent experiments. Above each graph is reported a representative western blot; β-actin was used as loading control. Arrowhead indicates an extra band recognized by the α-SG antibody that probably represents an immature form of the protein. ( B, E, H ) Cytotoxicity of correctors evaluated as the release of the cytosolic enzyme LDH in the culture medium of cells expressing R98H-α-SG treated with increasing concentration of C6 (B), D97G-α-SG treated with increasing concentration of C5 (E) or V247M-α-SG treated with increasing concentration of C17 (H). LDH release is expressed as percentage of the total amount of enzyme in cell lysate. ( C , F , I ) cell viability evaluated by measuring the metabolism of cells expressing R98H-α-SG treated with increasing concentration of C6 (C), D97G-α-SG treated with increasing concentration of C5 (F) or V247M-α-SG treated with increasing concentration of C17 (I). Cell viability was expressed as percentage (± SEM) toward cells treated with vehicle. Statistical analysis was performed by One-way ANOVA test - multiple comparisons Dunnett test; n.s., P > 0.05; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001.

    Journal: Human Molecular Genetics

    Article Title: Repairing folding-defective α-sarcoglycan mutants by CFTR correctors, a potential therapy for limb-girdle muscular dystrophy 2D

    doi: 10.1093/hmg/ddy013

    Figure Lengend Snippet: Corrector C6, C5 and C17 induced a dose-dependent increase of different α-SG mutants without major toxic effects. ( A , D , G ) Quantification of α-SG content in HEK293 cells expressing R98H-α-SG (A), D97G-α-SG (D) or V247M-α-SG (G) treated for 24 h with increasing concentrations of the indicated correctors. α-SG protein content was determined by WB and densitometric analysis on total protein lysates from at least three independent experiments. Above each graph is reported a representative western blot; β-actin was used as loading control. Arrowhead indicates an extra band recognized by the α-SG antibody that probably represents an immature form of the protein. ( B, E, H ) Cytotoxicity of correctors evaluated as the release of the cytosolic enzyme LDH in the culture medium of cells expressing R98H-α-SG treated with increasing concentration of C6 (B), D97G-α-SG treated with increasing concentration of C5 (E) or V247M-α-SG treated with increasing concentration of C17 (H). LDH release is expressed as percentage of the total amount of enzyme in cell lysate. ( C , F , I ) cell viability evaluated by measuring the metabolism of cells expressing R98H-α-SG treated with increasing concentration of C6 (C), D97G-α-SG treated with increasing concentration of C5 (F) or V247M-α-SG treated with increasing concentration of C17 (I). Cell viability was expressed as percentage (± SEM) toward cells treated with vehicle. Statistical analysis was performed by One-way ANOVA test - multiple comparisons Dunnett test; n.s., P > 0.05; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001.

    Article Snippet: Antibodies Mouse monoclonal antibody specific for α-SG (NCL-a-SARC) was from Leica Biosystem; rabbit monoclonal anti α-SG (AB189254) was from Abcam; mouse monoclonal antibody specific for β-SG, δ-SG, γ-SG and β-actin were from Sigma, rabbit polyclonal antibody specific for α-and δ-SG were produced as previously described , rabbit polyclonal antibody specific for α-actinin was from Santa Cruz.

    Techniques: Expressing, Western Blot, Concentration Assay

    Isolation of alpha-sarcoglycan + EVs from plasma using immunoaffinity capturing. Anti-alpha-sarcoglycan antibodies were conjugated to magnetic beads to isolate muscle EVs from plasma. Western blot analysis confirmed the presence of the exosomal marker Tsg101 in isolated EVs. Ponceau S Staining has been used as loading control (a). MicroRNA quantifications showed an increase in the miR-206/miR-16 ratio in the SGCA + sub-population of EVs (SGCA-Beads) compared to total (total plasma EVs) or uncaptured EVs (Supernatant). MiR-206 expression levels were normalized versus the endogenous reference miR-16 and expressed as -ΔCq (where ΔCq = Cq miR-206 -Cq miR-16 ) (b). Moreover, the quantification of miR-16 ratio in the SGCA + sub-population of EVs compared to total or uncaptured EVs shows that SGCA-conjugated beads retained about 2–5% of the total amount of EVs, miR-16 expression levels were normalized versus the spike-in reference cel-miR-39 and expressed as -ΔCq (where ΔCq = Cq miR-16 -Cq cel-miR-39 ) (c). Asterisks denote significant changes ( p

    Journal: PLoS ONE

    Article Title: Muscle Releases Alpha-Sarcoglycan Positive Extracellular Vesicles Carrying miRNAs in the Bloodstream

    doi: 10.1371/journal.pone.0125094

    Figure Lengend Snippet: Isolation of alpha-sarcoglycan + EVs from plasma using immunoaffinity capturing. Anti-alpha-sarcoglycan antibodies were conjugated to magnetic beads to isolate muscle EVs from plasma. Western blot analysis confirmed the presence of the exosomal marker Tsg101 in isolated EVs. Ponceau S Staining has been used as loading control (a). MicroRNA quantifications showed an increase in the miR-206/miR-16 ratio in the SGCA + sub-population of EVs (SGCA-Beads) compared to total (total plasma EVs) or uncaptured EVs (Supernatant). MiR-206 expression levels were normalized versus the endogenous reference miR-16 and expressed as -ΔCq (where ΔCq = Cq miR-206 -Cq miR-16 ) (b). Moreover, the quantification of miR-16 ratio in the SGCA + sub-population of EVs compared to total or uncaptured EVs shows that SGCA-conjugated beads retained about 2–5% of the total amount of EVs, miR-16 expression levels were normalized versus the spike-in reference cel-miR-39 and expressed as -ΔCq (where ΔCq = Cq miR-16 -Cq cel-miR-39 ) (c). Asterisks denote significant changes ( p

    Article Snippet: Isolation of exosomes using alpha-sarcoglycan immunoaffinity capture beads Exosome-Dynabeads Streptavidin (Life technologies) in PBS + 0.1% BSA were mixed with biotinylated anti-alpha-sarcoglycan (clone AD1/20A6 Monosan) according to the manufacturer’s instructions.

    Techniques: Isolation, Magnetic Beads, Western Blot, Marker, Staining, Expressing

    The muscle biopsy of the patient with the genetically confirmed LGMD2D showing no sign of dystrophy (A) and normal expression of all sarcoglycans (B, C, D, E) . The biopsy was taken at the age of 8 years.

    Journal: Acta Myologica

    Article Title: Childhood onset limb-girdle muscular dystrophies in the Aegean part of Turkey

    doi:

    Figure Lengend Snippet: The muscle biopsy of the patient with the genetically confirmed LGMD2D showing no sign of dystrophy (A) and normal expression of all sarcoglycans (B, C, D, E) . The biopsy was taken at the age of 8 years.

    Article Snippet: Spectrin (Novo-castra, UK, NCL-spec1), dystrophin N-terminus (Novo-castra, UK, NCL-dys3), adhalin (Novocastra, UK, NCL-a-sarc), other sarcoglycans (beta, delta, gamma; Novo-castra, UK, NCL-b-d-g-sarc), laminin alpha-2 chain (Novo-castra, UK, NCL-merosin), myotilin (Novo-castra, UK, NCL-myotilin), collagen VI (Novocastra, UK, NCL-COLL-vI), β-dystroglycan (Novocastra, UK, NCL-b-DG), HLA Class 1 (Novo-castra, UK, NCL-HLA-ABC), NCAM (ThermoScientific, CA, USA, CD56), nitric oxide synthase-1 (Novo-castra, UK, NCLNOS-1), emerin (Novo-castra, UK, NCL-emerin), caveolin 3 (Novus Biologicals, CA, USA, NB110-5029), calpain 3 (Abcam, Cambridge, UK, ab103250) and dysferlin (Novo-castra, UK, NCL-Hamlet-2) antibodies were used by standard techniques for immunohistochemical analyses.

    Techniques: Expressing

    Dantrolene/e23AON Combination Therapy Restores Sarcolemmal Dystrophin and DGC after Chronic Treatment Effect of dantrolene/e23AON combination therapy on rescue of DGC components as illustrated by representative immunofluorescence images of serial cross-sections from treated mdx quadriceps. Dystrophin was detected with an antibody specific to the rod domain (MANDYS8). Additional DGC components, α-sarcoglycan and β-dystroglycan, were detected with NCL-α-SARC and NCL-β-DG antibodies, respectively. ‡ Dantrolene was dosed at 30–70 mg/kg/day.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Repurposing Dantrolene for Long-Term Combination Therapy to Potentiate Antisense-Mediated DMD Exon Skipping in the mdx Mouse

    doi: 10.1016/j.omtn.2018.02.002

    Figure Lengend Snippet: Dantrolene/e23AON Combination Therapy Restores Sarcolemmal Dystrophin and DGC after Chronic Treatment Effect of dantrolene/e23AON combination therapy on rescue of DGC components as illustrated by representative immunofluorescence images of serial cross-sections from treated mdx quadriceps. Dystrophin was detected with an antibody specific to the rod domain (MANDYS8). Additional DGC components, α-sarcoglycan and β-dystroglycan, were detected with NCL-α-SARC and NCL-β-DG antibodies, respectively. ‡ Dantrolene was dosed at 30–70 mg/kg/day.

    Article Snippet: IHC assessment used the following primary antibodies: MANDYS8 (dystrophin rod domain), Ab15277 (dystrophin C terminus; Abcam), and Manex1A (dystrophin N terminus; Developmental Studies Hybridoma Bank). α-Sarcoglycan was detected with NCL-α-SARC (Novocastra), and β-dystroglycan was detected with NCL-β-DG (Novocastra), eMHC (DSHB; BF-G6-c), and DNA with DAPI.

    Techniques: Immunofluorescence

    Immunostaining for α-sarcoglycan and α-dystroglycan in cardiomyocytes. (a), Representative photomicrographs (×600, scale bar = 50μm) of immunostaining of α-sarcoglycan and α-dystroglycan in cardiomyocytes. (b), Quantitative analysis of immunostaining showed significantly increased staining of both α-sarcoglycan and α-dystroglycan in the HMGB1 group than the control group. HMGB1, high-mobility group box 1.

    Journal: PLoS ONE

    Article Title: The administration of high-mobility group box 1 fragment prevents deterioration of cardiac performance by enhancement of bone marrow mesenchymal stem cell homing in the delta-sarcoglycan-deficient hamster

    doi: 10.1371/journal.pone.0202838

    Figure Lengend Snippet: Immunostaining for α-sarcoglycan and α-dystroglycan in cardiomyocytes. (a), Representative photomicrographs (×600, scale bar = 50μm) of immunostaining of α-sarcoglycan and α-dystroglycan in cardiomyocytes. (b), Quantitative analysis of immunostaining showed significantly increased staining of both α-sarcoglycan and α-dystroglycan in the HMGB1 group than the control group. HMGB1, high-mobility group box 1.

    Article Snippet: The paraffin sections were used for immunohistochemistry and labeled using polyclonal CD31 antibody (1:50 CD31, Abcam, Cambridge, UK), anti-α-sarcoglycan (clone: Ad1/20A6; Novocastra, Weltzar, Germany), and anti-α-dystroglycan (clone: VIA4-1; Upstate Biotechnology, Lake Placid, NY) to assess capillary vascular density and the organization of cytoskeletal proteins.

    Techniques: Immunostaining, Staining

    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

    Reduced α- and γ- sarcoglycan expression in immature biglycan null mice Immunohistochemical analysis of P14, A. , and P21, B. , mouse muscle. Sections of quadriceps femoris from congenic P14 wild type and biglycan null (KO) mice were sectioned, mounted on the same slides and immunolabelled for dystrophin or α-, γ-, or δ- sarcoglycan. A. α-Sarcoglycan levels at the sarcolemma of P14 biglycan null muscle are selectively reduced in the biglycan null as compared to wild type muscle. B. γ-Sarcoglycan expression is reduced in P21 biglycan null mice, while α-sarcoglycan is unchanged. The level of dystrophin and δ-sarcoglycan is the same at both ages. Scale bars = 10 μm. C. Biochemical analysis of sarcoglycan expression. α- and γ- Sarcoglycan expression is reduced at distinct postnatal ages in skeletal muscle membranes from immature mice. KCl-washed skeletal muscle membranes (3 μg) from P14 or P21 congenic wild type or biglycan null mice (KO) were separated via SDS-PAGE. Western blotting was performed for α-, γ-, or δ- sarcoglycan or actin (loading control). Western blotting for α- and γ- sarcoglycan was performed on the same gel. Western blotting for δ-sarcoglycan and actin was performed in parallel on the same gel. α- Sarcoglycan levels are reduced in P14 biglycan null membranes while γ-sarcoglycan levels are reduced in P21 biglycan null membranes. Equivalent levels of δ-sarcoglycan and actin expression are seen at both ages. Similar results were observed in muscles from two other sets of mice.

    Journal: Journal of cellular physiology

    Article Title: Biglycan binds to ?- and ?- sarcoglycan and regulates their expression during development

    doi: 10.1002/jcp.20740

    Figure Lengend Snippet: Reduced α- and γ- sarcoglycan expression in immature biglycan null mice Immunohistochemical analysis of P14, A. , and P21, B. , mouse muscle. Sections of quadriceps femoris from congenic P14 wild type and biglycan null (KO) mice were sectioned, mounted on the same slides and immunolabelled for dystrophin or α-, γ-, or δ- sarcoglycan. A. α-Sarcoglycan levels at the sarcolemma of P14 biglycan null muscle are selectively reduced in the biglycan null as compared to wild type muscle. B. γ-Sarcoglycan expression is reduced in P21 biglycan null mice, while α-sarcoglycan is unchanged. The level of dystrophin and δ-sarcoglycan is the same at both ages. Scale bars = 10 μm. C. Biochemical analysis of sarcoglycan expression. α- and γ- Sarcoglycan expression is reduced at distinct postnatal ages in skeletal muscle membranes from immature mice. KCl-washed skeletal muscle membranes (3 μg) from P14 or P21 congenic wild type or biglycan null mice (KO) were separated via SDS-PAGE. Western blotting was performed for α-, γ-, or δ- sarcoglycan or actin (loading control). Western blotting for α- and γ- sarcoglycan was performed on the same gel. Western blotting for δ-sarcoglycan and actin was performed in parallel on the same gel. α- Sarcoglycan levels are reduced in P14 biglycan null membranes while γ-sarcoglycan levels are reduced in P21 biglycan null membranes. Equivalent levels of δ-sarcoglycan and actin expression are seen at both ages. Similar results were observed in muscles from two other sets of mice.

    Article Snippet: Immunoprecipitation of digitonin solubilized skeletal muscle membrane fractions with anti-dystrophin (anti 6-10) or anti-sarcoglycan antibodies (NovoCastra) were performed using the Seize × Immunoprecipitation kit (Pierce, Rockford, IL).

    Techniques: Expressing, Mouse Assay, Immunohistochemistry, SDS Page, Western Blot

    The biglycan core polypeptide is sufficient for binding to both immobilized and soluble α- and γ-sarcoglycan A. Purified recombinant biglycan core polypeptide (1μg) was separated by SDS-PAGE and either silver stained or blotted and probed as described above. α-Dystroglycan did not bind to this GAG-free biglycan. In contrast, both α- and γ- sarcoglycan bind to the biglycan core polypeptide. B. Co-immunoprecipitation of purified recombinant biglycan to recombinant sarcoglycans. His-tagged biglycan core polypeptide (0.5μg/ml) was incubated with the indicated 35 S-methionine-labeled, in vitro translated sarcoglycan for 1 hr followed by either anti-biglycan (a), anti-poly-His (b) or normal rabbit Ig (c). Immune complexes were then precipitated with protein G beads and analyzed by SDS-PAGE and autoradiography. Note that both α- and γ- sarcoglycan co-immunoprecipitate with biglycan, while β- and δ- sarcoglycan do not. The labelling of the various sarcoglycans is shown by direct autoradiography of SDS-PAGE-separated in vitro translated polypeptides (‘Input’).

    Journal: Journal of cellular physiology

    Article Title: Biglycan binds to ?- and ?- sarcoglycan and regulates their expression during development

    doi: 10.1002/jcp.20740

    Figure Lengend Snippet: The biglycan core polypeptide is sufficient for binding to both immobilized and soluble α- and γ-sarcoglycan A. Purified recombinant biglycan core polypeptide (1μg) was separated by SDS-PAGE and either silver stained or blotted and probed as described above. α-Dystroglycan did not bind to this GAG-free biglycan. In contrast, both α- and γ- sarcoglycan bind to the biglycan core polypeptide. B. Co-immunoprecipitation of purified recombinant biglycan to recombinant sarcoglycans. His-tagged biglycan core polypeptide (0.5μg/ml) was incubated with the indicated 35 S-methionine-labeled, in vitro translated sarcoglycan for 1 hr followed by either anti-biglycan (a), anti-poly-His (b) or normal rabbit Ig (c). Immune complexes were then precipitated with protein G beads and analyzed by SDS-PAGE and autoradiography. Note that both α- and γ- sarcoglycan co-immunoprecipitate with biglycan, while β- and δ- sarcoglycan do not. The labelling of the various sarcoglycans is shown by direct autoradiography of SDS-PAGE-separated in vitro translated polypeptides (‘Input’).

    Article Snippet: Immunoprecipitation of digitonin solubilized skeletal muscle membrane fractions with anti-dystrophin (anti 6-10) or anti-sarcoglycan antibodies (NovoCastra) were performed using the Seize × Immunoprecipitation kit (Pierce, Rockford, IL).

    Techniques: Binding Assay, Purification, Recombinant, SDS Page, Staining, Immunoprecipitation, Incubation, Labeling, In Vitro, Autoradiography

    Distinct binding sites for α- and γ- sarcoglycan on the biglycan core polypeptide A. Domain structure of biglycan, decorin and a biglycan-decorin chimera. The location of the pre-pro peptide (‘prepro’), 6-His tag, cysteine-rich amino- and carboxyl- domains, LRRs (ten open rectangles in the central domain; some schemes predict an 11 th in the carboxyl-terminal cysteine-rich region) and GAG attachment sites (asterisks) are indicated. Note that these sites are present in the recombinant proteins used in this experiment, but they are not substituted with GAGs. B. Binding of sarcoglycans to biglycan, decorin and a chimera. One microgram of each of the purified recombinant proteins was separated by SDS-PAGE and either directly stained (‘silver’) or blotted and probed with 35 S-methionine-labelled, in vitro-translated sarcoglycans as indicated. Both α- and γ- sarcoglycan bind to the immobilized biglycan core but not to decorin core. In contrast, only α-sarcoglycan binds to the biglycan-decorin chimeric protein. Thus the first 30 amino acids of biglycan is necessary for its binding to α-sarcoglycan. Neither β- nor δ- sarcoglycan bind to biglycan, decorin or the chimera. C. Competition studies. Sarcoglycan binding to purified recombinant biglycan core polypeptide in the presence of excess MBP-Bgn 38-77 (amino acids 38 to 77 of biglycan) or MBP-Dcn 31-71 (amino acids 31 to 71 of decorin). These sequences correspond to the first 40 amino acids of the mature biglycan and decorin polypeptides, respectively. Four micrograms of biglycan were separated by SDS-PAGE and either directly stained with Coomassie Blue (CB) or blotted and probed with biotinylated (BT), in vitro-translated α- or γ-sarcoglycan as indicated. Binding of α-sarcoglycan to full-length biglycan was inhibited in the presence of MBP-Bgn 38-77 , while binding between γ-sarcoglycan and biglycan binding remained unchanged. The decorin fusion protein did not inhibit this interaction.

    Journal: Journal of cellular physiology

    Article Title: Biglycan binds to ?- and ?- sarcoglycan and regulates their expression during development

    doi: 10.1002/jcp.20740

    Figure Lengend Snippet: Distinct binding sites for α- and γ- sarcoglycan on the biglycan core polypeptide A. Domain structure of biglycan, decorin and a biglycan-decorin chimera. The location of the pre-pro peptide (‘prepro’), 6-His tag, cysteine-rich amino- and carboxyl- domains, LRRs (ten open rectangles in the central domain; some schemes predict an 11 th in the carboxyl-terminal cysteine-rich region) and GAG attachment sites (asterisks) are indicated. Note that these sites are present in the recombinant proteins used in this experiment, but they are not substituted with GAGs. B. Binding of sarcoglycans to biglycan, decorin and a chimera. One microgram of each of the purified recombinant proteins was separated by SDS-PAGE and either directly stained (‘silver’) or blotted and probed with 35 S-methionine-labelled, in vitro-translated sarcoglycans as indicated. Both α- and γ- sarcoglycan bind to the immobilized biglycan core but not to decorin core. In contrast, only α-sarcoglycan binds to the biglycan-decorin chimeric protein. Thus the first 30 amino acids of biglycan is necessary for its binding to α-sarcoglycan. Neither β- nor δ- sarcoglycan bind to biglycan, decorin or the chimera. C. Competition studies. Sarcoglycan binding to purified recombinant biglycan core polypeptide in the presence of excess MBP-Bgn 38-77 (amino acids 38 to 77 of biglycan) or MBP-Dcn 31-71 (amino acids 31 to 71 of decorin). These sequences correspond to the first 40 amino acids of the mature biglycan and decorin polypeptides, respectively. Four micrograms of biglycan were separated by SDS-PAGE and either directly stained with Coomassie Blue (CB) or blotted and probed with biotinylated (BT), in vitro-translated α- or γ-sarcoglycan as indicated. Binding of α-sarcoglycan to full-length biglycan was inhibited in the presence of MBP-Bgn 38-77 , while binding between γ-sarcoglycan and biglycan binding remained unchanged. The decorin fusion protein did not inhibit this interaction.

    Article Snippet: Immunoprecipitation of digitonin solubilized skeletal muscle membrane fractions with anti-dystrophin (anti 6-10) or anti-sarcoglycan antibodies (NovoCastra) were performed using the Seize × Immunoprecipitation kit (Pierce, Rockford, IL).

    Techniques: Binding Assay, Recombinant, Purification, SDS Page, Staining, In Vitro

    Biglycan binds to α- and γ- sarcoglycan A. Sarcoglycan binding to native biglycan. Postsynaptic membrane fractions from Torpedo electric organ (TEOM; 0.8 μg) were separated on SDS-PAGE gels, blotted onto nitrocellulose then probed with either 35 ). No binding of β– or δ– sarcoglycan to this or any other polypeptide in these fractions was detected. B. Binding of α-dystroglycan and sarcoglycans to purified recombinant biglycan proteoglycan (Biglycan-PG). One microgram of biglycan was separated by SDS-PAGE and either stained with silver or blotted onto nitrocellulose (‘Overlay’) and probed as described above. α-Dystroglycan and α- and γ-sarcoglycan bind to this recombinant, GAG-containing biglycan proteoglycan, while no binding of β– or δ– sarcoglycan is detected.

    Journal: Journal of cellular physiology

    Article Title: Biglycan binds to ?- and ?- sarcoglycan and regulates their expression during development

    doi: 10.1002/jcp.20740

    Figure Lengend Snippet: Biglycan binds to α- and γ- sarcoglycan A. Sarcoglycan binding to native biglycan. Postsynaptic membrane fractions from Torpedo electric organ (TEOM; 0.8 μg) were separated on SDS-PAGE gels, blotted onto nitrocellulose then probed with either 35 ). No binding of β– or δ– sarcoglycan to this or any other polypeptide in these fractions was detected. B. Binding of α-dystroglycan and sarcoglycans to purified recombinant biglycan proteoglycan (Biglycan-PG). One microgram of biglycan was separated by SDS-PAGE and either stained with silver or blotted onto nitrocellulose (‘Overlay’) and probed as described above. α-Dystroglycan and α- and γ-sarcoglycan bind to this recombinant, GAG-containing biglycan proteoglycan, while no binding of β– or δ– sarcoglycan is detected.

    Article Snippet: Immunoprecipitation of digitonin solubilized skeletal muscle membrane fractions with anti-dystrophin (anti 6-10) or anti-sarcoglycan antibodies (NovoCastra) were performed using the Seize × Immunoprecipitation kit (Pierce, Rockford, IL).

    Techniques: Binding Assay, SDS Page, Purification, Recombinant, Staining

    AAV-mediated microdystrophin expression restored DGC. A, Mock-treated and AAV-infected mdx hearts and age-matched BL10 heart were evaluated with monoclonal antibodies specific to N-terminal (human-specific) and C-terminal of dystrophin, β -sarcoglycan, and β -dystroglycan. Nuclei were counterstained with DAPI. Bar=100 μ m. B, Serial sections of virus-specific microdystrophin expression and recruitment of β -sarcoglycan and β -dystroglycan. DIC indicates differential interference contrast. Bar=200 μ m.

    Journal: Circulation

    Article Title: Microdystrophin Gene Therapy of Cardiomyopathy Restores Dystrophin-Glycoprotein Complex and Improves Sarcolemma Integrity in the Mdx Mouse Heart

    doi: 10.1161/01.CIR.0000089371.11664.27

    Figure Lengend Snippet: AAV-mediated microdystrophin expression restored DGC. A, Mock-treated and AAV-infected mdx hearts and age-matched BL10 heart were evaluated with monoclonal antibodies specific to N-terminal (human-specific) and C-terminal of dystrophin, β -sarcoglycan, and β -dystroglycan. Nuclei were counterstained with DAPI. Bar=100 μ m. B, Serial sections of virus-specific microdystrophin expression and recruitment of β -sarcoglycan and β -dystroglycan. DIC indicates differential interference contrast. Bar=200 μ m.

    Article Snippet: Additional monoclonal antibodies, including anti–dystrophin-C-terminal Dys-2 (1:30), anti– β -sarcoglycan (1:50), and anti– β -dystroglycan (1:50) antibodies (Novocastra) were used the same way as for Dys-3.

    Techniques: Expressing, Infection

    Reduction of dystrophin, dystroglycan and β-sarcoglycan mRNAs in synthetic smooth muscle cells. Relative amounts of dystrophin, dystroglycan and β-sarcoglycan mRNAs in contractile and synthetic smooth muscle cells (n = 6 and 8, respectively). The GAPDH gene expression served as a reference. ***, p

    Journal: PLoS ONE

    Article Title: Increased Neointimal Thickening in Dystrophin-Deficient mdx Mice

    doi: 10.1371/journal.pone.0029904

    Figure Lengend Snippet: Reduction of dystrophin, dystroglycan and β-sarcoglycan mRNAs in synthetic smooth muscle cells. Relative amounts of dystrophin, dystroglycan and β-sarcoglycan mRNAs in contractile and synthetic smooth muscle cells (n = 6 and 8, respectively). The GAPDH gene expression served as a reference. ***, p

    Article Snippet: Antibodies against β-dystroglycan were described previously ; smooth muscle α-actin antibodies (clone 1A4) were from Sigma; anti-β-sarcoglycan antibodies (clone βSarc/5B1) from Novocastra; anti-CD68 antibodies (clone FA-11) from AbD Serotec and anti-PCNA antiserum from Abcam (ab15497).

    Techniques: Expressing

    Impaired sarcolemmal glycoprotein trafficking in hearts overexpressing secretion-defective Thbs4. (A) Western blotting for membrane glycoproteins purified from hearts of transgenic mice overexpressing wild-type Thbs4 (DTG-Thbs4) or the secretion-defective mutant (DTG-mCa 2+ ). Cadherin was used as a loading control. (B) Representative experiment of immunocytochemistry for β-sarcoglycan and the Golgi marker GM130 in cardiomyocytes isolated from transgenic hearts of the indicated genotype. Scale bar = 50 μm. Similar results were obtained in 2 additional experiments.

    Journal: Molecular and Cellular Biology

    Article Title: Defective Flux of Thrombospondin-4 through the Secretory Pathway Impairs Cardiomyocyte Membrane Stability and Causes Cardiomyopathy

    doi: 10.1128/MCB.00114-18

    Figure Lengend Snippet: Impaired sarcolemmal glycoprotein trafficking in hearts overexpressing secretion-defective Thbs4. (A) Western blotting for membrane glycoproteins purified from hearts of transgenic mice overexpressing wild-type Thbs4 (DTG-Thbs4) or the secretion-defective mutant (DTG-mCa 2+ ). Cadherin was used as a loading control. (B) Representative experiment of immunocytochemistry for β-sarcoglycan and the Golgi marker GM130 in cardiomyocytes isolated from transgenic hearts of the indicated genotype. Scale bar = 50 μm. Similar results were obtained in 2 additional experiments.

    Article Snippet: Proteins were resolved by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes (Millipore), and immunoblotted with antibodies for Flag (Cell Signaling Technology or Sigma), calreticulin, PDI, integrin β1, pan-cadherin, and Rab6 (Cell Signaling Technology), Armet, SAR1, δ-sarcoglycan, and β-actin (Abcam), α-sarcoglycan and β-dystroglycan (Developmental Studies Hybridoma Bank), β-sarcoglycan (Novus Biologicals), Rab24 (BD Transduction), atrial natriuretic factor (ANF; Millipore), α-tubulin, BiP, and α-sarcomeric actin (Sigma), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Fitzgerald), and Thbs4 (Santa Cruz).

    Techniques: Western Blot, Purification, Transgenic Assay, Mouse Assay, Mutagenesis, Immunocytochemistry, Marker, Isolation

    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

    Morphological and molecular evidences of myocardial hypertrophy progression. (A) Immunohistochemical staining of beta- sarcoglycan allowing to detect outer membrane showing clear increase of cells size in transverse orientation by 10-weeks group compared to intact. (B) Increase in cell diameter (Dmin) illustrating progressive cardiomyocytes enlargement, and significant increase after 8 and 10 weeks of aortic banding performing. Nppa expression was upregulated in LV (C) and IVS (D) after 1, 2, 8, and 10 weeks of model duration compared to intact or sham-control groups. (E) Positive linear correlation was found for Nppa mRNA level and left ventricular mass (LVM) indexed to body weight. The analysis included experimental groups after 8 and 10 weeks of aortic constriction, 8 week’s sham-operated and intact animals, r indicates Pearson coefficient; for all ∗ P

    Journal: Frontiers in Genetics

    Article Title: Time- and Ventricular-Specific Expression Profiles of Genes Encoding Z-Disk Proteins in Pressure Overload Model of Left Ventricular Hypertrophy

    doi: 10.3389/fgene.2018.00684

    Figure Lengend Snippet: Morphological and molecular evidences of myocardial hypertrophy progression. (A) Immunohistochemical staining of beta- sarcoglycan allowing to detect outer membrane showing clear increase of cells size in transverse orientation by 10-weeks group compared to intact. (B) Increase in cell diameter (Dmin) illustrating progressive cardiomyocytes enlargement, and significant increase after 8 and 10 weeks of aortic banding performing. Nppa expression was upregulated in LV (C) and IVS (D) after 1, 2, 8, and 10 weeks of model duration compared to intact or sham-control groups. (E) Positive linear correlation was found for Nppa mRNA level and left ventricular mass (LVM) indexed to body weight. The analysis included experimental groups after 8 and 10 weeks of aortic constriction, 8 week’s sham-operated and intact animals, r indicates Pearson coefficient; for all ∗ P

    Article Snippet: After blocking in 15% fetal calf serum (FCS) for 30 min at room temperature, samples were incubated overnight at +4°C with mouse monoclonal beta-sarcoglycan antibodies (Leica Biosystems, NCL-L-b-SARC).

    Techniques: Immunohistochemistry, Staining, Expressing

    Adenoviral gene transfer efficiency and expression of wild-type (WT) and mutant δ-sarcoglycan (δSG) in adult cardiac myocytes. A : δSG adenoviral vector design. B : time course of δSG expression using adenoviral vectors in

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: Dilated cardiomyopathy mutations in δ-sarcoglycan exert a dominant-negative effect on cardiac myocyte mechanical stability

    doi: 10.1152/ajpheart.00521.2015

    Figure Lengend Snippet: Adenoviral gene transfer efficiency and expression of wild-type (WT) and mutant δ-sarcoglycan (δSG) in adult cardiac myocytes. A : δSG adenoviral vector design. B : time course of δSG expression using adenoviral vectors in

    Article Snippet: Primary antibodies included: A-14 rabbit polyclonal anti-myc (Santa Cruz Biotechnology; 1:100), goat anti-human δ-sarcoglycan (R & D Systems; 1:1,000), IIH6 anti-α dystroglycan (Millipore; 1:1,000), and mouse anti-β dystroglycan (Vector Laboratories; 1:1,000).

    Techniques: Expressing, Mutagenesis, Plasmid Preparation

    Functional and phenotypic correction in mdx mice following treatments with PNA AOs at 100 mg/kg for five weekly intravenous injections . ( a ) Serial immunostaining of dystrophin-associated protein complex (DAPC) in mdx mice. DAPC protein components β-dystroglycan, α-sarcoglycan, and β-sarcoglycan and neuronal nitric oxide synthase (nNOS) were detected in quadriceps from mdx mice treated with PNA AOs. Arrowheads point to the identical muscle fibers (bar = 200 μm). ( b ) Muscle function was assessed using grip strength test to determine the physical improvement of PNA AOs treated mdx mice. Significant force recovery was detected in treated mdx mice compared with untreated mdx controls ( t -test, * P

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Peptide Nucleic Acid Promotes Systemic Dystrophin Expression and Functional Rescue in Dystrophin-deficient mdx Mice

    doi: 10.1038/mtna.2015.27

    Figure Lengend Snippet: Functional and phenotypic correction in mdx mice following treatments with PNA AOs at 100 mg/kg for five weekly intravenous injections . ( a ) Serial immunostaining of dystrophin-associated protein complex (DAPC) in mdx mice. DAPC protein components β-dystroglycan, α-sarcoglycan, and β-sarcoglycan and neuronal nitric oxide synthase (nNOS) were detected in quadriceps from mdx mice treated with PNA AOs. Arrowheads point to the identical muscle fibers (bar = 200 μm). ( b ) Muscle function was assessed using grip strength test to determine the physical improvement of PNA AOs treated mdx mice. Significant force recovery was detected in treated mdx mice compared with untreated mdx controls ( t -test, * P

    Article Snippet: Rabbit polyclonal antibody to neuronal nitric oxide synthase and mouse monoclonal antibodies to β-dystroglycan, α-sarcoglycan, and β-sarcoglycan were used according to manufacturer's instructions (Novocastra, Newcastle upon Tyne, UK).

    Techniques: Functional Assay, Mouse Assay, Immunostaining

    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: Dystrophin, utrophin, α-dystroglycan, α-Sarcoglycan, γ-Sarcoglycan and δ-Sarcoglycan were detected using the antibodies described earlier. β-Sarcoglycan was revealed with a mouse monoclonal antibody against the β-sarcoglycan N-terminus (NCL-b-SARC, 1:250; clone 5B1, IgG1; Novocastra).

    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: Dystrophin, utrophin, α-dystroglycan, α-Sarcoglycan, γ-Sarcoglycan and δ-Sarcoglycan were detected using the antibodies described earlier. β-Sarcoglycan was revealed with a mouse monoclonal antibody against the β-sarcoglycan N-terminus (NCL-b-SARC, 1:250; clone 5B1, IgG1; Novocastra).

    Techniques: Double Knockout, Mouse Assay

    HDAC4 directly represses structural gene expression via binding and inhibition of MEF2 activity. A ) Promoter sequence of γ-sarcoglycan spanning −812 to −861 nt upstream of transcriptional start. AT-rich MEF2 sites are indicated above, which were mutated singly in or combination for subsequent luciferase assays. B ) C2C12 myoblasts were tranfected with luciferase reporter constructs containing wild-type or MEF2 binding site deletion (ΔAT1, ΔAT2, or ΔAT1,2) promoters as indicated. Transfections occurred in the presence of MEF2 and/or HDAC4 as indicated. Luciferase activity was determined 48 h after transfection. C . D ) ChIP analysis was performed on C2C12 myotubes using either control IgG-, MEF2-, or HDAC4-specific antibodies. Bound DNA was analyzed by standard PCR generating a 300 bp fragment encompassing both MEF2 binding sites in the γ-sarcoglycan promoter. Input lane represents 5% of total starting material.

    Journal: The FASEB Journal

    Article Title: The deacetylase HDAC4 controls myocyte enhancing factor-2-dependent structural gene expression in response to neural activity

    doi: 10.1096/fj.08-115931

    Figure Lengend Snippet: HDAC4 directly represses structural gene expression via binding and inhibition of MEF2 activity. A ) Promoter sequence of γ-sarcoglycan spanning −812 to −861 nt upstream of transcriptional start. AT-rich MEF2 sites are indicated above, which were mutated singly in or combination for subsequent luciferase assays. B ) C2C12 myoblasts were tranfected with luciferase reporter constructs containing wild-type or MEF2 binding site deletion (ΔAT1, ΔAT2, or ΔAT1,2) promoters as indicated. Transfections occurred in the presence of MEF2 and/or HDAC4 as indicated. Luciferase activity was determined 48 h after transfection. C . D ) ChIP analysis was performed on C2C12 myotubes using either control IgG-, MEF2-, or HDAC4-specific antibodies. Bound DNA was analyzed by standard PCR generating a 300 bp fragment encompassing both MEF2 binding sites in the γ-sarcoglycan promoter. Input lane represents 5% of total starting material.

    Article Snippet: The following muscle specific antibodies were used: dystrophin (DYS1), β,γ-sarcoglycan, and desmin (Novocastra, Newcastle upon Tyne, UK); monoclonal myogenin antibody (Sigma, St. Louis, MO, USA); monoclonal caveolin-3 antibody (BD Biosciences, San Jose, CA, USA); and myotilin antibody (a kind gift from Mike Hauser, Duke University, Durham, NC, USA; ref. ).

    Techniques: Expressing, Binding Assay, Inhibition, Activity Assay, Sequencing, Luciferase, Construct, Transfection, Chromatin Immunoprecipitation, Polymerase Chain Reaction

    β, γ, δ, and ε sarcoglycan are not overexpressed in Galgt2 transgenic Sgca −/− muscle. A : Immunostaining of gastrocnemius muscle in Galgt2 transgenic (CT) and non-transgenic Sgca +/− and Sgca −/− mice. β, γ, and ε sarcoglycan were not increased in expression along myofibers in Sgca −/− CT muscle, while ( B ) immunostaining of α dystroglycan, β dystroglycan, dystrophin, and utrophin was high in Sgca +/− CT and Sgca −/− CT muscle. Scale bar =100 μm ( A and B ). C : 40 μg of SDS whole muscle lysate from gastrocnemius is loaded per lane. β, γ and δ sarcoglycan protein levels were not increased in Sgca −/− CT muscle as they were in Sgca +/− CT muscle. Utrophin, α dystroglycan, and plectin 1, by contrast, were similarly increased in both Sgca −/− CT and Sgca +/− CT muscle. Actin is shown as a control for protein loading and transfer.

    Journal: The American Journal of Pathology

    Article Title: Overexpression of Galgt2 Reduces Dystrophic Pathology in the Skeletal Muscles of Alpha Sarcoglycan-Deficient Mice

    doi: 10.2353/ajpath.2009.080967

    Figure Lengend Snippet: β, γ, δ, and ε sarcoglycan are not overexpressed in Galgt2 transgenic Sgca −/− muscle. A : Immunostaining of gastrocnemius muscle in Galgt2 transgenic (CT) and non-transgenic Sgca +/− and Sgca −/− mice. β, γ, and ε sarcoglycan were not increased in expression along myofibers in Sgca −/− CT muscle, while ( B ) immunostaining of α dystroglycan, β dystroglycan, dystrophin, and utrophin was high in Sgca +/− CT and Sgca −/− CT muscle. Scale bar =100 μm ( A and B ). C : 40 μg of SDS whole muscle lysate from gastrocnemius is loaded per lane. β, γ and δ sarcoglycan protein levels were not increased in Sgca −/− CT muscle as they were in Sgca +/− CT muscle. Utrophin, α dystroglycan, and plectin 1, by contrast, were similarly increased in both Sgca −/− CT and Sgca +/− CT muscle. Actin is shown as a control for protein loading and transfer.

    Article Snippet: A rabbit polyclonal antiserum to ε sarcoglycan (H-67) and a mouse monoclonal antibody to Plectin 1 (10F6) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Transgenic Assay, Immunostaining, Mouse Assay, Expressing

    Creation of Galgt2 transgenic α sarcoglycan-deficient ( Sgca −/− ) mice. A : Galgt2 transgenic (CT) mice and non-transgenic littermates that were either heterozygous ( Sgca +/− ) or homozygous ( Sgca −/− ) for a deletion of α sarcoglycan were stained with a monoclonal antibody to α sarcoglycan or with CT2, an antibody that recognizes the CT carbohydrate. Control secondary antibody for CT2 (IgM only) and α sarcoglycan (IgG only) are also shown. Scale bar = 100 μm. B : 40 μg of whole muscle SDS protein lysate was blotted for Galgt2 protein and for α sarcoglycan protein. Actin was used as a control for protein loading and transfer. Asterisk indicates native molecular weight for the protein of interest. C : Sgca +/− CT and Sgca −/− CT muscles show similar increases in Galgt2 mRNA, relative to Sgca +/− and Sgca −/− , respectively, as measured by quantitative RT-PCR. D : Galgt2 mRNA was reduced in Sgca −/− muscles relative to Sgca +/− . Gas, gastrocnemius; Quad, quadriceps; TA, tibialis anterior; Tri, triceps; Dia, diaphragm. Errors are SD for 3 to 4 animals per condition (in C and D ).

    Journal: The American Journal of Pathology

    Article Title: Overexpression of Galgt2 Reduces Dystrophic Pathology in the Skeletal Muscles of Alpha Sarcoglycan-Deficient Mice

    doi: 10.2353/ajpath.2009.080967

    Figure Lengend Snippet: Creation of Galgt2 transgenic α sarcoglycan-deficient ( Sgca −/− ) mice. A : Galgt2 transgenic (CT) mice and non-transgenic littermates that were either heterozygous ( Sgca +/− ) or homozygous ( Sgca −/− ) for a deletion of α sarcoglycan were stained with a monoclonal antibody to α sarcoglycan or with CT2, an antibody that recognizes the CT carbohydrate. Control secondary antibody for CT2 (IgM only) and α sarcoglycan (IgG only) are also shown. Scale bar = 100 μm. B : 40 μg of whole muscle SDS protein lysate was blotted for Galgt2 protein and for α sarcoglycan protein. Actin was used as a control for protein loading and transfer. Asterisk indicates native molecular weight for the protein of interest. C : Sgca +/− CT and Sgca −/− CT muscles show similar increases in Galgt2 mRNA, relative to Sgca +/− and Sgca −/− , respectively, as measured by quantitative RT-PCR. D : Galgt2 mRNA was reduced in Sgca −/− muscles relative to Sgca +/− . Gas, gastrocnemius; Quad, quadriceps; TA, tibialis anterior; Tri, triceps; Dia, diaphragm. Errors are SD for 3 to 4 animals per condition (in C and D ).

    Article Snippet: A rabbit polyclonal antiserum to ε sarcoglycan (H-67) and a mouse monoclonal antibody to Plectin 1 (10F6) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Transgenic Assay, Mouse Assay, Staining, Molecular Weight, Quantitative RT-PCR

    Paramagnetic Bead Conjugation With Cell-Specific Antibodies Conjugation of green fluorescent microspheres with γ-sarcoglycan antibody (red fluorescence) after activation of the beads’ surface carboxyl groups by carbodiimide (EDAC) (A, top ) . In the absence of EDAC, no antibody conjugation was seen (A, bottom ). (B) Schematic representation of conjugation of NRVMs with γ-sarcoglycan antibody coated microspheres.

    Journal: Journal of the American College of Cardiology

    Article Title: Engineered Electrical Conduction Tract Restores Conduction in Complete Heart Block: From In Vitro to In Vivo Proof-of-Concept

    doi: 10.1016/j.jacc.2014.09.056

    Figure Lengend Snippet: Paramagnetic Bead Conjugation With Cell-Specific Antibodies Conjugation of green fluorescent microspheres with γ-sarcoglycan antibody (red fluorescence) after activation of the beads’ surface carboxyl groups by carbodiimide (EDAC) (A, top ) . In the absence of EDAC, no antibody conjugation was seen (A, bottom ). (B) Schematic representation of conjugation of NRVMs with γ-sarcoglycan antibody coated microspheres.

    Article Snippet: Paramagnetic fluorescent microspheres (8 μm diameter, Bangs Laboratories, Fishers, IN) were conjugated with γ-sarcoglycan antibody (200 μg; Santa Cruz Biotechnology, Dallas, TX) or with CD105 antibody (200 μg; R & D Technologies, North Kingstown, RI) using the Polylink Protein Coupling Kit (Bangs Laboratories) as per the manufacturer’s instructions.

    Techniques: Conjugation Assay, Fluorescence, Activation Assay

    Loss of HSPB7 results in FLNC and γ-sarcoglycan protein aggregation. Confocal micrographs of longitudinal sections (A) and cross-sections (B,C) of the diaphragm of 12-week-old mice. Specific antibodies were used to identify the distributions of the sarcomeric components α-actinin (Z-line), FLNC and γ-sarcoglycan as indicated. FLNC (arrows in A,C) and γ-sarcoglycan (arrows in B) aggregation were observed in the diaphragm muscle. The arrangement of α-actinin was normal (arrowheads in A). However, Z-line disarray was also observed in the diaphragm of CKO mice (asterisks in A). FLNC and γ-sarcoglycan were discontinuous or missing in some regions of the sarcolemma (arrowheads in B,C). Extracellular matrix accumulation was labeled with wheat germ agglutinin (WGA). FLNC mislocalizes on the sarcolemma (arrowheads in C). The nucleus was visualized by Hoechst 33342 staining. (D) Western blot analysis of expression levels of FLNC and α-actinin in the diaphragm muscle. The muscle homogenate supernatant (S) and pellet (P) fractions from control (Flox/Flox) and CKO mice at 12 and 36 weeks of age. GAPDH was used to verify the loading amount in the supernatant. Coomassie Blue staining (45–75 kDa) was used to verify the loading amount in the pellet. The graph shows the mean±s.d. densitometry results ( n =3). * P

    Journal: Journal of Cell Science

    Article Title: HSPB7 interacts with dimerized FLNC and its absence results in progressive myopathy in skeletal muscles

    doi: 10.1242/jcs.179887

    Figure Lengend Snippet: Loss of HSPB7 results in FLNC and γ-sarcoglycan protein aggregation. Confocal micrographs of longitudinal sections (A) and cross-sections (B,C) of the diaphragm of 12-week-old mice. Specific antibodies were used to identify the distributions of the sarcomeric components α-actinin (Z-line), FLNC and γ-sarcoglycan as indicated. FLNC (arrows in A,C) and γ-sarcoglycan (arrows in B) aggregation were observed in the diaphragm muscle. The arrangement of α-actinin was normal (arrowheads in A). However, Z-line disarray was also observed in the diaphragm of CKO mice (asterisks in A). FLNC and γ-sarcoglycan were discontinuous or missing in some regions of the sarcolemma (arrowheads in B,C). Extracellular matrix accumulation was labeled with wheat germ agglutinin (WGA). FLNC mislocalizes on the sarcolemma (arrowheads in C). The nucleus was visualized by Hoechst 33342 staining. (D) Western blot analysis of expression levels of FLNC and α-actinin in the diaphragm muscle. The muscle homogenate supernatant (S) and pellet (P) fractions from control (Flox/Flox) and CKO mice at 12 and 36 weeks of age. GAPDH was used to verify the loading amount in the supernatant. Coomassie Blue staining (45–75 kDa) was used to verify the loading amount in the pellet. The graph shows the mean±s.d. densitometry results ( n =3). * P

    Article Snippet: The primary antibodies used were: mouse monoclonal anti-α-actinin (clone EA-53, Sigma-Aldrich; 1:1000), anti-GAPDH (clone 6C5, Millipore Corporation; 1:3000), anti-MHC [MF20, Developmental Studies Hybridoma Bank (DSHB), 1:1000], anti-myogenin (anti-MyoG; F5D, DSHB; 1:1000) antibodies; rabbit polyclonal anti-FLAG (F7425, Sigma-Aldrich; 1:3000), anti-Myc (LTK BioLaboratories; 1:3000), anti-HA (Abcam; 1:3000), anti-γ-sarcoglycan (H-60, Santa Cruz Biotechnology; 1:200), anti-MyoD (M-318, Santa Cruz Biotechnology; 1:1000) and anti-dystrophin (ab15277, Abcam; 1:1000) antibodies; goat polyclonal anti-FLNC (K-18, Santa Cruz Biotechnology; 1:1000) antibodies and guinea pig polyclonal antibody against the NH2-terminal of the murine HSPB7 peptide (amino acids 29–84, LTK BioLaboratories).

    Techniques: Mouse Assay, Labeling, Whole Genome Amplification, Staining, Western Blot, Expressing

    Expression and localization of HSPB7 in skeletal muscle. (A) Western blot analysis of calf muscle showing HSPB7 expression from embryonic stages to adulthood (E14.5 to P56). (B) Immunohistochemical analysis of HSPB7 and MHC expression at E13.5 (arrows, muscle tissue; arrowheads, heart). (C) Western blot analysis of HSPB7 expression in the heart and various skeletal muscles. GAPDH was used as loading control. EDL, extensor digitorum longorum; TA, tibialis anterior. (D) Subcellular localization of HSPB7 in the soleus muscle of adult mice. The muscle sections were co-immunostained with antibodies against HSPB7 (red) and desmin (green), sarcomeric α-actinin (green) or γ-sarcoglycan (green). The nucleus was visualized by Hoechst 33342 staining. The arrows in A and C represent the expression of HSPB7. Scale bars: 500 μm (B, upper and middle panel); 200 μm (B, lower panel); 10 μm (D).

    Journal: Journal of Cell Science

    Article Title: HSPB7 interacts with dimerized FLNC and its absence results in progressive myopathy in skeletal muscles

    doi: 10.1242/jcs.179887

    Figure Lengend Snippet: Expression and localization of HSPB7 in skeletal muscle. (A) Western blot analysis of calf muscle showing HSPB7 expression from embryonic stages to adulthood (E14.5 to P56). (B) Immunohistochemical analysis of HSPB7 and MHC expression at E13.5 (arrows, muscle tissue; arrowheads, heart). (C) Western blot analysis of HSPB7 expression in the heart and various skeletal muscles. GAPDH was used as loading control. EDL, extensor digitorum longorum; TA, tibialis anterior. (D) Subcellular localization of HSPB7 in the soleus muscle of adult mice. The muscle sections were co-immunostained with antibodies against HSPB7 (red) and desmin (green), sarcomeric α-actinin (green) or γ-sarcoglycan (green). The nucleus was visualized by Hoechst 33342 staining. The arrows in A and C represent the expression of HSPB7. Scale bars: 500 μm (B, upper and middle panel); 200 μm (B, lower panel); 10 μm (D).

    Article Snippet: The primary antibodies used were: mouse monoclonal anti-α-actinin (clone EA-53, Sigma-Aldrich; 1:1000), anti-GAPDH (clone 6C5, Millipore Corporation; 1:3000), anti-MHC [MF20, Developmental Studies Hybridoma Bank (DSHB), 1:1000], anti-myogenin (anti-MyoG; F5D, DSHB; 1:1000) antibodies; rabbit polyclonal anti-FLAG (F7425, Sigma-Aldrich; 1:3000), anti-Myc (LTK BioLaboratories; 1:3000), anti-HA (Abcam; 1:3000), anti-γ-sarcoglycan (H-60, Santa Cruz Biotechnology; 1:200), anti-MyoD (M-318, Santa Cruz Biotechnology; 1:1000) and anti-dystrophin (ab15277, Abcam; 1:1000) antibodies; goat polyclonal anti-FLNC (K-18, Santa Cruz Biotechnology; 1:1000) antibodies and guinea pig polyclonal antibody against the NH2-terminal of the murine HSPB7 peptide (amino acids 29–84, LTK BioLaboratories).

    Techniques: Expressing, Western Blot, Immunohistochemistry, Mouse Assay, Staining