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Abcam rabbit monoclonal anti α sg
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.
Rabbit Monoclonal Anti α Sg, supplied by Abcam, used in various techniques. Bioz Stars score: 96/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 96 stars, based on 3 article reviews
Price from $9.99 to $1999.99
rabbit monoclonal anti α sg - by Bioz Stars, 2022-10
96/100 stars

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1) Product Images from "Repairing folding-defective α-sarcoglycan mutants by CFTR correctors, a potential therapy for limb-girdle muscular dystrophy 2D"

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

Journal: Human Molecular Genetics

doi: 10.1093/hmg/ddy013

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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: Expressing, Western Blot, Concentration Assay

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    Abcam anti tollip antibody
    <t>Tollip</t> is required for the recruitment of NBR1, TAX1BP1, and NDP52 to GAS-containing LC3 vacuoles. (A) Wild-type and Tollip-knockout HeLa cells were transfected with EmGFP-LC3 or co-transfected with EmGFP-LC3 and mCherry-NDP52. After infecting with GAS for 4 h, endogenous NBR1, TAX1BP1, and p62 were labeled with <t>antibodies.</t> Insets: enlarged boxed areas; top: EmGFP-LC3; middle: autophagy receptors; bottom: merged image. All images are representative of 3 independent experiments. Scale bar, 10 μm. (B) Percentages of recruitment of autophagy receptors to GAS-containing autophagosome-like vacuoles: NBR1, TAX1BP1, p62, and mCherry-NDP52 (from left to right). In each experiment, > 50 LC3-positive vacuoles were evaluated per sample; bars: means ± SD of 3 independent experiments. *P
    Anti Tollip Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Abcam rabbit monoclonal anti α sg
    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.
    Rabbit Monoclonal Anti α Sg, 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
    https://www.bioz.com/result/rabbit monoclonal anti α sg/product/Abcam
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    rabbit monoclonal anti α sg - by Bioz Stars, 2022-10
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    Abcam rabbit polyclonal anti β sg
    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.
    Rabbit Polyclonal Anti β Sg, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Tollip is required for the recruitment of NBR1, TAX1BP1, and NDP52 to GAS-containing LC3 vacuoles. (A) Wild-type and Tollip-knockout HeLa cells were transfected with EmGFP-LC3 or co-transfected with EmGFP-LC3 and mCherry-NDP52. After infecting with GAS for 4 h, endogenous NBR1, TAX1BP1, and p62 were labeled with antibodies. Insets: enlarged boxed areas; top: EmGFP-LC3; middle: autophagy receptors; bottom: merged image. All images are representative of 3 independent experiments. Scale bar, 10 μm. (B) Percentages of recruitment of autophagy receptors to GAS-containing autophagosome-like vacuoles: NBR1, TAX1BP1, p62, and mCherry-NDP52 (from left to right). In each experiment, > 50 LC3-positive vacuoles were evaluated per sample; bars: means ± SD of 3 independent experiments. *P

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Autophagy Receptor Tollip Facilitates Bacterial Autophagy by Recruiting Galectin-7 in Response to Group A Streptococcus Infection

    doi: 10.3389/fcimb.2020.583137

    Figure Lengend Snippet: Tollip is required for the recruitment of NBR1, TAX1BP1, and NDP52 to GAS-containing LC3 vacuoles. (A) Wild-type and Tollip-knockout HeLa cells were transfected with EmGFP-LC3 or co-transfected with EmGFP-LC3 and mCherry-NDP52. After infecting with GAS for 4 h, endogenous NBR1, TAX1BP1, and p62 were labeled with antibodies. Insets: enlarged boxed areas; top: EmGFP-LC3; middle: autophagy receptors; bottom: merged image. All images are representative of 3 independent experiments. Scale bar, 10 μm. (B) Percentages of recruitment of autophagy receptors to GAS-containing autophagosome-like vacuoles: NBR1, TAX1BP1, p62, and mCherry-NDP52 (from left to right). In each experiment, > 50 LC3-positive vacuoles were evaluated per sample; bars: means ± SD of 3 independent experiments. *P

    Article Snippet: The following primary and secondary antibodies were used in the study: mouse monoclonal anti-galectin-3 (1:250, B2C10; BD Biosciences, 556904), rabbit polyclonal anti-galectin-7 (1:250 dilution for immunofluorescence, 1:1000 dilution for Western blot, Abcam, ab10482), mouse monoclonal anti-LAMP-1 (1:250, H4A3; Santa Cruz Biotechnology, sc-20011), rabbit polyclonal anti-Tollip (1:1000, Abcam, ab187198), rabbit monoclonal anti-NBR1 (1;250, D2E6; Cell Signaling, 9891), mouse monoclonal anti-p62 (1:250, D-3; Santa Cruz Biotechnology, sc-28359), rabbit monoclonal anti-TAX1BP1 (1:250, D1D5; Cell Signaling, 5105), mouse monoclonal anti-streptolysin (1:2000, 6D11; Abcam, ab23501), mouse monoclonal anti-GFP (1:1000, GF200; Nacalai Tesque, 04363‐24), mouse monoclonal anti-GAPDH (1:1000, 6C5; Santa Cruz Biotechnology, sc-32233), and mouse monoclonal anti-FLAG (1:1000, M2; Sigma-Aldrich, A2220).

    Techniques: Knock-Out, Transfection, Labeling

    Tollip is required for autophagosome and autophagolysosome formation during GAS infection. (A) Tollip-knockout (KO) cells were generated using CRISPR/Cas9-mediated gene editing. Immunoblotting was used to detect Tollip protein expression in wild-type (WT) and Tollip-knockout cells. (B) HeLa cells transfected with EmGFP-LC3 were infected with GAS, and at 2 hpi, immunofluorescence analysis was performed to examine the formation of bacterium-containing autophagosomes. (C) Cells harboring GAS-containing autophagosomes (left) and cells containing LC3-positive fragments (right) were manually counted using immunofluorescence confocal microscopy. In each experiment, > 100 autophagosomes were evaluated per sample; bars: means ± SD of 3 independent experiments. P values less than 0.05 were considered to indicate statistical significance, and are marked * for P

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Autophagy Receptor Tollip Facilitates Bacterial Autophagy by Recruiting Galectin-7 in Response to Group A Streptococcus Infection

    doi: 10.3389/fcimb.2020.583137

    Figure Lengend Snippet: Tollip is required for autophagosome and autophagolysosome formation during GAS infection. (A) Tollip-knockout (KO) cells were generated using CRISPR/Cas9-mediated gene editing. Immunoblotting was used to detect Tollip protein expression in wild-type (WT) and Tollip-knockout cells. (B) HeLa cells transfected with EmGFP-LC3 were infected with GAS, and at 2 hpi, immunofluorescence analysis was performed to examine the formation of bacterium-containing autophagosomes. (C) Cells harboring GAS-containing autophagosomes (left) and cells containing LC3-positive fragments (right) were manually counted using immunofluorescence confocal microscopy. In each experiment, > 100 autophagosomes were evaluated per sample; bars: means ± SD of 3 independent experiments. P values less than 0.05 were considered to indicate statistical significance, and are marked * for P

    Article Snippet: The following primary and secondary antibodies were used in the study: mouse monoclonal anti-galectin-3 (1:250, B2C10; BD Biosciences, 556904), rabbit polyclonal anti-galectin-7 (1:250 dilution for immunofluorescence, 1:1000 dilution for Western blot, Abcam, ab10482), mouse monoclonal anti-LAMP-1 (1:250, H4A3; Santa Cruz Biotechnology, sc-20011), rabbit polyclonal anti-Tollip (1:1000, Abcam, ab187198), rabbit monoclonal anti-NBR1 (1;250, D2E6; Cell Signaling, 9891), mouse monoclonal anti-p62 (1:250, D-3; Santa Cruz Biotechnology, sc-28359), rabbit monoclonal anti-TAX1BP1 (1:250, D1D5; Cell Signaling, 5105), mouse monoclonal anti-streptolysin (1:2000, 6D11; Abcam, ab23501), mouse monoclonal anti-GFP (1:1000, GF200; Nacalai Tesque, 04363‐24), mouse monoclonal anti-GAPDH (1:1000, 6C5; Santa Cruz Biotechnology, sc-32233), and mouse monoclonal anti-FLAG (1:1000, M2; Sigma-Aldrich, A2220).

    Techniques: Infection, Knock-Out, Generated, CRISPR, Expressing, Transfection, Immunofluorescence, Confocal Microscopy

    Tollip is involved in the recruitment of galectin-1 and -7 to GAS-containing LC3-vacuoles. (A) Wild-type and Tollip-knockout HeLa cells transfected with EmGFP-galectin-7 and mCherry-LC3 were infected with GAS; at 4 hpi, immunofluorescence analysis was performed to examine the localization of the expressed proteins. (B) EmGFP-galectin-positive autophagosomes were manually counted using immunofluorescence confocal microscopy. In each experiment, > 50 GAS-containing LC3-vacuoles were counted and evaluated per sample; bars: means ± SD of 3 independent experiments. (C) Wild-type and Tollip-knockout HeLa cells transfected with EmGFP-LC3 were infected with GAS; at 4 hpi, immunofluorescence analysis was used to examine the localization of endogenous galectin-1 and EmGFP-LC3; galectin-1 was labeled using a rabbit anti-galectin-1 antibody. (D) Cells containing galectin-1-positive GAS-containing autophagosomes were manually counted using immunofluorescence confocal microscopy. In each experiment, > 50 GAS-containing autophagosomes were counted and evaluated per sample; bars: means ± SD of 3 independent experiments. (E) Wild-type and Tollip-knockout HeLa cells were infected with GAS; at 4 hpi, immunofluorescence analysis was performed to examine the SLO protein expression from GAS. Scale bar, 10 μm. (F) Cells containing SLO-positive GAS were manually counted using immunofluorescence confocal microscopy. In each experiment, > 200 GAS-infected cells were evaluated per sample; bars: means ± SD of 3 independent experiments. (G) Tollip interacts with galectin-7. HeLa cells were co-transfected with EmGFP-galectin-7 and FLAG-Tollip for 48 h, after which immunoprecipitation was performed using anti-FLAG. The immunoprecipitates were immunoblotted with anti-GFP to detect EmGFP-galectin-7. Experiments were repeated more than three times. *P

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Autophagy Receptor Tollip Facilitates Bacterial Autophagy by Recruiting Galectin-7 in Response to Group A Streptococcus Infection

    doi: 10.3389/fcimb.2020.583137

    Figure Lengend Snippet: Tollip is involved in the recruitment of galectin-1 and -7 to GAS-containing LC3-vacuoles. (A) Wild-type and Tollip-knockout HeLa cells transfected with EmGFP-galectin-7 and mCherry-LC3 were infected with GAS; at 4 hpi, immunofluorescence analysis was performed to examine the localization of the expressed proteins. (B) EmGFP-galectin-positive autophagosomes were manually counted using immunofluorescence confocal microscopy. In each experiment, > 50 GAS-containing LC3-vacuoles were counted and evaluated per sample; bars: means ± SD of 3 independent experiments. (C) Wild-type and Tollip-knockout HeLa cells transfected with EmGFP-LC3 were infected with GAS; at 4 hpi, immunofluorescence analysis was used to examine the localization of endogenous galectin-1 and EmGFP-LC3; galectin-1 was labeled using a rabbit anti-galectin-1 antibody. (D) Cells containing galectin-1-positive GAS-containing autophagosomes were manually counted using immunofluorescence confocal microscopy. In each experiment, > 50 GAS-containing autophagosomes were counted and evaluated per sample; bars: means ± SD of 3 independent experiments. (E) Wild-type and Tollip-knockout HeLa cells were infected with GAS; at 4 hpi, immunofluorescence analysis was performed to examine the SLO protein expression from GAS. Scale bar, 10 μm. (F) Cells containing SLO-positive GAS were manually counted using immunofluorescence confocal microscopy. In each experiment, > 200 GAS-infected cells were evaluated per sample; bars: means ± SD of 3 independent experiments. (G) Tollip interacts with galectin-7. HeLa cells were co-transfected with EmGFP-galectin-7 and FLAG-Tollip for 48 h, after which immunoprecipitation was performed using anti-FLAG. The immunoprecipitates were immunoblotted with anti-GFP to detect EmGFP-galectin-7. Experiments were repeated more than three times. *P

    Article Snippet: The following primary and secondary antibodies were used in the study: mouse monoclonal anti-galectin-3 (1:250, B2C10; BD Biosciences, 556904), rabbit polyclonal anti-galectin-7 (1:250 dilution for immunofluorescence, 1:1000 dilution for Western blot, Abcam, ab10482), mouse monoclonal anti-LAMP-1 (1:250, H4A3; Santa Cruz Biotechnology, sc-20011), rabbit polyclonal anti-Tollip (1:1000, Abcam, ab187198), rabbit monoclonal anti-NBR1 (1;250, D2E6; Cell Signaling, 9891), mouse monoclonal anti-p62 (1:250, D-3; Santa Cruz Biotechnology, sc-28359), rabbit monoclonal anti-TAX1BP1 (1:250, D1D5; Cell Signaling, 5105), mouse monoclonal anti-streptolysin (1:2000, 6D11; Abcam, ab23501), mouse monoclonal anti-GFP (1:1000, GF200; Nacalai Tesque, 04363‐24), mouse monoclonal anti-GAPDH (1:1000, 6C5; Santa Cruz Biotechnology, sc-32233), and mouse monoclonal anti-FLAG (1:1000, M2; Sigma-Aldrich, A2220).

    Techniques: Knock-Out, Transfection, Infection, Immunofluorescence, Confocal Microscopy, Labeling, Expressing, Immunoprecipitation

    Model depicting Tollip function in xenophagy. Tollip localizes on the inner surface of the cell membrane by binding to PtdIns 3P through its C2 domain. After GAS invasion through endocytosis, Tollip surrounds GAS before GAS damages the endosomal membrane and escapes into the cytosol. After GAS damages the endosomal membrane, galectin-1 and -7 are recruited to the membrane fragments and associate with Tollip, and then other autophagy receptors, including NBR1, TAX1BP1, p62, and NDP52, are recruited.

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Autophagy Receptor Tollip Facilitates Bacterial Autophagy by Recruiting Galectin-7 in Response to Group A Streptococcus Infection

    doi: 10.3389/fcimb.2020.583137

    Figure Lengend Snippet: Model depicting Tollip function in xenophagy. Tollip localizes on the inner surface of the cell membrane by binding to PtdIns 3P through its C2 domain. After GAS invasion through endocytosis, Tollip surrounds GAS before GAS damages the endosomal membrane and escapes into the cytosol. After GAS damages the endosomal membrane, galectin-1 and -7 are recruited to the membrane fragments and associate with Tollip, and then other autophagy receptors, including NBR1, TAX1BP1, p62, and NDP52, are recruited.

    Article Snippet: The following primary and secondary antibodies were used in the study: mouse monoclonal anti-galectin-3 (1:250, B2C10; BD Biosciences, 556904), rabbit polyclonal anti-galectin-7 (1:250 dilution for immunofluorescence, 1:1000 dilution for Western blot, Abcam, ab10482), mouse monoclonal anti-LAMP-1 (1:250, H4A3; Santa Cruz Biotechnology, sc-20011), rabbit polyclonal anti-Tollip (1:1000, Abcam, ab187198), rabbit monoclonal anti-NBR1 (1;250, D2E6; Cell Signaling, 9891), mouse monoclonal anti-p62 (1:250, D-3; Santa Cruz Biotechnology, sc-28359), rabbit monoclonal anti-TAX1BP1 (1:250, D1D5; Cell Signaling, 5105), mouse monoclonal anti-streptolysin (1:2000, 6D11; Abcam, ab23501), mouse monoclonal anti-GFP (1:1000, GF200; Nacalai Tesque, 04363‐24), mouse monoclonal anti-GAPDH (1:1000, 6C5; Santa Cruz Biotechnology, sc-32233), and mouse monoclonal anti-FLAG (1:1000, M2; Sigma-Aldrich, A2220).

    Techniques: Binding Assay

    Tollip is recruited to GAS-containing vacuoles before GAS damages the endosomal membrane. (A) HeLa cells transfected with mCherry-Tollip and EmGFP-LC3 were infected with Group A Streptococcus (GAS) for 4 h, and immunostained with anti-Group A Streptococcus carbohydrate (GAC) to stain GAS. Immunofluorescence analysis was performed to determine the localization of mCherry-Tollip and EmGFP-LC3. Cellular and bacterial DNA were stained with DAPI. Insets: enlarged boxed areas. All images are representative of at least 3 independent experiments. Scale bar, 10 μm. (B) Percentages of cells containing Tollip-positive GAS and cells containing Tollip-positive autophagosomes; cells were manually counted using immunofluorescence confocal microscopy. In each experiment, > 60 autophagosomes were evaluated per sample; bars: means ± SD of 3 independent experiments. (C) HeLa cells were transfected with mCherry-Tollip and infected with GAS or SLO-deletion-mutant (ΔSLO) GAS for 4 h, and then stained for endogenous galectin-3. Immunofluorescence microscopy was used to examine the localization of mCherry-Tollip and endogenous galectin-3. Insets: enlarged boxed areas. All images are representative of at least 3 independent experiments. Scale bar, 10 μm.

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Autophagy Receptor Tollip Facilitates Bacterial Autophagy by Recruiting Galectin-7 in Response to Group A Streptococcus Infection

    doi: 10.3389/fcimb.2020.583137

    Figure Lengend Snippet: Tollip is recruited to GAS-containing vacuoles before GAS damages the endosomal membrane. (A) HeLa cells transfected with mCherry-Tollip and EmGFP-LC3 were infected with Group A Streptococcus (GAS) for 4 h, and immunostained with anti-Group A Streptococcus carbohydrate (GAC) to stain GAS. Immunofluorescence analysis was performed to determine the localization of mCherry-Tollip and EmGFP-LC3. Cellular and bacterial DNA were stained with DAPI. Insets: enlarged boxed areas. All images are representative of at least 3 independent experiments. Scale bar, 10 μm. (B) Percentages of cells containing Tollip-positive GAS and cells containing Tollip-positive autophagosomes; cells were manually counted using immunofluorescence confocal microscopy. In each experiment, > 60 autophagosomes were evaluated per sample; bars: means ± SD of 3 independent experiments. (C) HeLa cells were transfected with mCherry-Tollip and infected with GAS or SLO-deletion-mutant (ΔSLO) GAS for 4 h, and then stained for endogenous galectin-3. Immunofluorescence microscopy was used to examine the localization of mCherry-Tollip and endogenous galectin-3. Insets: enlarged boxed areas. All images are representative of at least 3 independent experiments. Scale bar, 10 μm.

    Article Snippet: The following primary and secondary antibodies were used in the study: mouse monoclonal anti-galectin-3 (1:250, B2C10; BD Biosciences, 556904), rabbit polyclonal anti-galectin-7 (1:250 dilution for immunofluorescence, 1:1000 dilution for Western blot, Abcam, ab10482), mouse monoclonal anti-LAMP-1 (1:250, H4A3; Santa Cruz Biotechnology, sc-20011), rabbit polyclonal anti-Tollip (1:1000, Abcam, ab187198), rabbit monoclonal anti-NBR1 (1;250, D2E6; Cell Signaling, 9891), mouse monoclonal anti-p62 (1:250, D-3; Santa Cruz Biotechnology, sc-28359), rabbit monoclonal anti-TAX1BP1 (1:250, D1D5; Cell Signaling, 5105), mouse monoclonal anti-streptolysin (1:2000, 6D11; Abcam, ab23501), mouse monoclonal anti-GFP (1:1000, GF200; Nacalai Tesque, 04363‐24), mouse monoclonal anti-GAPDH (1:1000, 6C5; Santa Cruz Biotechnology, sc-32233), and mouse monoclonal anti-FLAG (1:1000, M2; Sigma-Aldrich, A2220).

    Techniques: Transfection, Infection, Staining, Immunofluorescence, Confocal Microscopy, Mutagenesis, Microscopy

    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