Standard western blotting protocol was used. Gastroctrocnemius muscle was lysed with ice-cold RIPA buffer in the presence of 1X protease inhibitor cocktail (Sigma). For each sample, 10 µg to 100 µg of total protein was loaded onto 4–12% SDS-polyacrylamide gradient gels (Invitrogen), and transferred to PVDF membranes. The membranes were blocked for 1 h with 1% BSA in PBST and incubated with various antibodies against dystrophin (Abcam), β-dystroglycan (β-DG) (Santa Cruz), caveolin-3 (BD Biosciences), dysferlin (Abcam), myosin (Iowa Hybridoma Bank), MG53 (Novus Biologicals), and Lamp1 (ID4B; Iowa Hybridoma Bank) in PBST. The blots were detected by using Peroxidase-conjugated anti-rabbit/mouse/rat secondary antibody with an enhanced chemiluminescence reagent (Amersham Pharmacia Biotech).
Caveolin 3
Manufactured by BD
Sourced in United States
Caveolin-3 is a membrane protein that plays a structural role in the formation of caveolae, invaginations of the plasma membrane. It is primarily expressed in skeletal and cardiac muscle cells.
Automatically generated - may contain errors
Lab products found in correlation
Dystrophin, by Abcam (2 mentions)
Dysferlin, by Abcam (2 mentions)
Gradient sds polyacrylamide gel, by Thermo Fisher Scientific (2 mentions)
β dystroglycan β dg, by Santa Cruz Biotechnology (2 mentions)
Peroxidase conjugated anti, by Cytiva (2 mentions)
Enhanced chemiluminescence reagent, by Cytiva (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Flotillin 2, by BD (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Goat anti rat hrp, by Jackson ImmunoResearch (1 mentions)
Mouse anti rabbit hrp, by Jackson ImmunoResearch (1 mentions)
Ha tag, by Roche (1 mentions)
Donkey anti guinea pig, by Jackson ImmunoResearch (1 mentions)
Goat anti rabbit hrp, by Jackson ImmunoResearch (1 mentions)
Protease phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Pp 1α, by Santa Cruz Biotechnology (1 mentions)
Troponin 1, by Cell Signaling Technology (1 mentions)
Pser 23 24 troponin 1, by Cell Signaling Technology (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
12 protocols using caveolin 3
1
Muscle Protein Expression Analysis
2
Muscle Protein Expression Analysis
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Standard western blotting protocol was used. Gastroctrocnemius muscle was lysed with ice-cold RIPA buffer in the presence of 1X protease inhibitor cocktail (Sigma). For each sample, 10 µg to 100 µg of total protein was loaded onto 4–12% SDS-polyacrylamide gradient gels (Invitrogen), and transferred to PVDF membranes. The membranes were blocked for 1 h with 1% BSA in PBST and incubated with various antibodies against dystrophin (Abcam), β-dystroglycan (β-DG) (Santa Cruz), caveolin-3 (BD Biosciences), dysferlin (Abcam), myosin (Iowa Hybridoma Bank), MG53 (Novus Biologicals), and Lamp1 (ID4B; Iowa Hybridoma Bank) in PBST. The blots were detected by using Peroxidase-conjugated anti-rabbit/mouse/rat secondary antibody with an enhanced chemiluminescence reagent (Amersham Pharmacia Biotech).
3
Western Blot Analysis of Membrane Proteins
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Standard western blotting was carried out using 6%–20% gradient gels. The primary antibodies used were as follows: zDHHC5 (1:1,000, Sigma, HPA014670), NCX1 (1:1,000, Swant, R3F1), PLM (FXYD1, 1:1,000, Abcam, ab76597), Flotillin-2 (1:1,000, BD Biosciences, 610383), Caveolin-3 (1:4,000, BD Biosciences, 610420), HA-tag (1:5,000, Roche, 11867423001). The secondary antibodies used were as follows: Rabbit anti-mouse HRP (1:2,000, Jackson ImmunoResearch 111-035-144), Goat anti-rabbit HRP (1:2,000, Jackson ImmunoResearch 315-035-003), Goat anti-rat HRP (1:2,000, Jackson ImmunoResearch, 313-035-003), Donkey anti-guinea pig (1:2,000, Jackson ImmunoResearch, 106-035-003).
4
Subcellular Fractionation and Protein Analysis
5
Protein Characterization by SDS-PAGE and Western Blot
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Protein separation by SDS-PAGE and western blot analysis were performed using methods that we have previously described. [27 (link)] Protein samples (10–40 μg) were separated and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). Ponceau S staining was used to verify equal protein loads. For the kinase studies membranes were probed with polyclonal antibodies recognizing dually phosphorylated forms of p38 (from Santa Cruz Biotechnology, Santa Cruz, CA), ERK1/2). Membranes were then stripped and blotted with polyclonal antibodies for p38α and ERK. Additional blots (10 μg protein) were probed with monoclonal antibodies for caveolin-3 (BD Pharmingen) and cytochrome-c oxidase (Invitrogen) or a polyclonal antibody for α-actinin (Santa Cruz). Bound antibodies were visualized by enhanced chemiluminescence (Amersham, Piscataway, NJ). Immunoreactive bands were quantified with UN-SCAN-IT gel digitizing software (Silk Scientific, Inc., Orem, UT).
6
HEK Cell Transfection and Western Blot Protocol
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All chemicals were of the highest grade available and were purchased from Sigma–Aldrich unless indicated otherwise. All HEK cell transfections utilized Lipofectamine 2000 (ThermoFisher Scientific). Primary antibodies were from the following sources: PLM phospho-S68—custom made38 (link), zDHHC5—Sigma–Aldrich HPA014670 (diluted 1:2000 for western blots), Flotillin 2—BD Biosciences 610383 clone 29 (1:2000), PLM—Abcam ab76597 (1:2000), HA tag—Roche, clone 3F10 (1:2000), FLAG—Sigma F1804 clone M2 (1:2000), Caveolin 3—BD Biosciences 610421 clone 26 (1:5000), sodium pump α1 subunit, Development Studies Hybridoma Bank, clone α6F (1:100), Ras—Merck 05-516, clone RAS10 (1:2000). HRP-linked secondary antibodies were from ThermoFisher Scientific (antirat, diluted 1:2000 for western blots) and Jackson ImmunoResearch (antirabbit and antimouse, both diluted 1:2000 for western blots). Western Blots utilized Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore, Watford, UK) and were visualized and analyzed using a ChemiDoc XRS acquisition system running QuantityOne software (BioRad).
7
Immunofluorescence Staining of Muscle Cells
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Cells were grown on Lab-TEK II™(Fisher Scientific) according to the previously explained protocol. Throughout the procedure, cells were kept at room temperature. After treatment, cells were fixed with 4% paraformaldehyde for 10 minutes and then washed in PBS for 10 minutes. Cells were then incubated for 10 minutes with a permeabilization solution (200 μL of PBS 1X +0.5% triton X-100 + protease inhibitors cocktail) (Roche). From there, cells were exposed to a blocking buffer (PBS+ 1% BSA + protease inhibitors cocktail) for 30 minutes. The primary antibody was applied in blocking buffer for 3 hours at room temperature, followed by one wash in PBS for 10 minutes and 1 hour of contact with the secondary antibody in blocking buffer. After a wash in PBS for 10 minutes, cells were fixed using a 4% paraformaldehyde solution in PBS for 10 minutes, then washed again. Finally, coverslips were mounted with Vectashield-Dapi 25 ng/mL, and kept at 4°C until pictures were taken. Dysferlin was detected using NCL-Hamlet at a dilution of 1:200. Caveolin-3 (BD Biosciences) was detected using a dilution of 1:1,000, and desmin (Fischer Scientific) using a dilution of 1:100. Scale bars are indicated on each picture.
Observation was performed using a Zeiss apotome microscope, and images were processed withAxioVision software and/or ImageJ software.
Observation was performed using a Zeiss apotome microscope, and images were processed withAxioVision software and/or ImageJ software.
8
Muscle Biopsy Analysis Protocol for Diagnostic Purposes
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All patients underwent muscle biopsy for diagnostic purposes at the time of their first evaluation in our center. Muscle biopsies were processed according to standard procedures, and routine histologic stains were analyzed.14 (link) Immunofluorescence was performed on 6-μm-thick sections using FITC-conjugated phalloidin (Sigma, St. Louis, MO) and the following primary antibodies: monoclonal anti-human CD4, CD8, CD20, CD68, and C5b9 antigens (Sigma, St. Louis, MO), and type I and II HLA (US Biological, Swampscott, MA), dystrophin (N-terminal and C-terminal from Novocastra/Leica Biosystems, Germany; rod domain from Merck-Millipore, MA), alpha-sarcoglycan and gamma-sarcoglycan (Novocastra/Leica Biosystems, Germany), alpha-dystroglycan (Merck-Millipore, MA), merosin (Merck-Millipore, MA), and caveolin-3 (BD Biosciences, CA).
9
Protein Sample Preparation and Immunoblotting
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Protein samples were prepared as previously described.
10 (link),
13 (link),
14 (link),
31 (link),
32 (link) Ventricular tissue fragments were disrupted with a PYREX® Potter‐Elvehjem tissue grinder on ice in lysis buffer containing proteinase and phosphatase inhibitors (Roche, Indianapolis, IN, USA). The homogenate was centrifuged at 15,000 g at 4°C for 15 min, and the supernatant was saved for immunoblotting. Total protein extracts were run in equal protein amount on SDS‐PAGE electrophoresis. The blots were probed with primary antibodies to ROCK1 (sc‐5560), ROCK2 (sc‐5561), Gαq (E‐17) (sc‐393), β1‐adrenergic receptor (β1‐AR) (sc‐568), adenylyl cyclase V/VI (AC5/6) (sc‐590) from Santa Cruz Biotechnology (Dallas, TX, USA), caveolin‐1 (#610407), caveolin‐2 (#610685), caveolin‐3 (#610421) from BD Biosciences (San Jose, CA, USA), ROCK1 (#4035), insulin receptor (IR) β (#3025), p‐Akt‐Ser473 (#9271), p‐Akt‐Thr308 (#9275), Akt (#9272), p‐GSK‐3β‐Ser9 (#9336) from Cell Signaling Technology (Danvers, MA, USA), p‐IR‐Tyr972 (#44800G) from Invitrogen. All blots were normalized to GAPDH (ABS16; MilliporeSigma, Burlington, MA, USA) or to actin (MABT523; MilliporeSigma).
10 (link),
13 (link),
14 (link),
31 (link),
32 (link) Ventricular tissue fragments were disrupted with a PYREX® Potter‐Elvehjem tissue grinder on ice in lysis buffer containing proteinase and phosphatase inhibitors (Roche, Indianapolis, IN, USA). The homogenate was centrifuged at 15,000 g at 4°C for 15 min, and the supernatant was saved for immunoblotting. Total protein extracts were run in equal protein amount on SDS‐PAGE electrophoresis. The blots were probed with primary antibodies to ROCK1 (sc‐5560), ROCK2 (sc‐5561), Gαq (E‐17) (sc‐393), β1‐adrenergic receptor (β1‐AR) (sc‐568), adenylyl cyclase V/VI (AC5/6) (sc‐590) from Santa Cruz Biotechnology (Dallas, TX, USA), caveolin‐1 (#610407), caveolin‐2 (#610685), caveolin‐3 (#610421) from BD Biosciences (San Jose, CA, USA), ROCK1 (#4035), insulin receptor (IR) β (#3025), p‐Akt‐Ser473 (#9271), p‐Akt‐Thr308 (#9275), Akt (#9272), p‐GSK‐3β‐Ser9 (#9336) from Cell Signaling Technology (Danvers, MA, USA), p‐IR‐Tyr972 (#44800G) from Invitrogen. All blots were normalized to GAPDH (ABS16; MilliporeSigma, Burlington, MA, USA) or to actin (MABT523; MilliporeSigma).
10
Immunohistochemistry of Cardiac Tissue
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For immunohistochemistry experiments, different hearts were dissected into left and right ventricles and fixed by immersion in 1% paraformaldehyde (w/v) in PBS at 4°C for 1 h. Fixed tissue was then washed and cryoprotected by immersing it through a series of sucrose solutions (series of 10, 20 and 30% w/v). Excess sucrose solution was removed before a thin layer of O.C.T. compound (Tissue-Tek) was applied to coat the tissue. The tissue was snap-frozen for 2 min by immersion in methylbutane (Sigma) within a container of liquid nitrogen. Frozen tissue blocks were cryosectioned with a Feather Blade at −20°C. Ten microlitre-thick sections were obtained and attached to coverslips until immunofluorescent labelling.
Cardiac tissue sections were treated with Image-iT FX signal enhancer (Thermo Fisher Scientific) for 1 h at room temperature (20–22°C) prior to incubation with primary antibodies (RyR: MA3-916 (Thermo Fisher Scientific); sodium–calcium exchanger: R3F1 (Swant); caveolin-3: 610420 (BD Transduction)), overnight at 4°C, and diluted 1 : 200 in incubation solution. After washing in PBS, sections were then incubated with secondary antibodies (as above) for 2 h at room temperature, in incubation solution.
Cardiac tissue sections were treated with Image-iT FX signal enhancer (Thermo Fisher Scientific) for 1 h at room temperature (20–22°C) prior to incubation with primary antibodies (RyR: MA3-916 (Thermo Fisher Scientific); sodium–calcium exchanger: R3F1 (Swant); caveolin-3: 610420 (BD Transduction)), overnight at 4°C, and diluted 1 : 200 in incubation solution. After washing in PBS, sections were then incubated with secondary antibodies (as above) for 2 h at room temperature, in incubation solution.
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