anti alpha sarcoglycan antibody epr14773 Search Results


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    Abcam anti alpha sarcoglycan antibody epr14773
    Corrector C17 induced a dose dependent increase of <t>α-SG</t> 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.
    Anti Alpha Sarcoglycan Antibody Epr14773, supplied by Abcam, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    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