SDS-PAGE was conducted on 3–15% linear gradient gel. 15-µg microsomes from HEK293 cells were applied to the gel. The separated proteins were electrophoretically transferred onto PVDF membranes. Western blotting was performed using antibodies for RyR2 (Chugun et al., 2003 (link)) and calnexin (C4731; Sigma-Aldrich). All of WT-, R2473S-, and K4750Q-expressing cells exhibited similar expression level of RyR2 proteins in ER membranes (Fig. S1 A).
C4731
Manufactured by Merck Group
Sourced in United States, United Kingdom
The C4731 is a laboratory equipment product manufactured by the Merck Group. It serves as a core component for various scientific applications. The device's primary function is to facilitate precise measurements and data collection within controlled laboratory environments. Detailed specifications and intended uses are not provided to maintain an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ultracut s, by Leica camera (1 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Merck Group (1 mentions)
Supersignal west pico kit, by Thermo Fisher Scientific (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
A6154, by Merck Group (1 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
Chemidoc xrs molecular imaging system, by Bio-Rad (1 mentions)
Hpa016480, by Merck Group (1 mentions)
Hybond ecl nitrocellulose membrane, by Cytiva (1 mentions)
M8159, by Merck Group (1 mentions)
Chemidoc mp system, by Bio-Rad (1 mentions)
Ncl dys2, by Leica Biosystems (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
0.2 m pore size nitrocellulose membrane, by Bio-Rad (1 mentions)
Mab901, by R&D Systems (1 mentions)
Af3074, by R&D Systems (1 mentions)
Ab155668, by Abcam (1 mentions)
Ab105767, by Abcam (1 mentions)
Sc 53540, by Santa Cruz Biotechnology (1 mentions)
18 protocols using c4731
1
Quantitative Evaluation of RyR2 Expression
2
Microscopic Tissue Preparation and Imaging
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For outer segment measurement studies, tissue was prepared as previously described (23 (link)). Semi-thin and ultra-thin sections were cut with a Leica Ultracut S microtome fitted with an appropriate type of diamond knife. For light microscopy semi-thin sections were stained with 1% toluidine blue and 1% borax in 50% ethanol. Ultra-thin sections were treated for 5 min with 1% uranyl acetate in 50% ethanol followed by Reynold’s lead citrate, prior to viewing and photography with a JEOL 1010 TEM operating at 80 kV.
For fluorescence studies, eyes were collected at specified time points and fixed overnight in 4% paraformaldehyde at 4 °C. Post-fixation eyes were cryoprotected by incubation in 30% sucrose in PBS. Eyes were then frozen and cyrosectioned as previously described (22 (link)). Cryosectioned eyes were stained with mouse anti-Rho-1D4 (1:1500), rabbit anti-calnexin (1:600; C4731, Sigma Aldrich) and rat anti-prominin-1 (1:200; clone 13A4, CD133, eBioscience) primary antibodies in blocking solution (3% BSA, 10% normal goat serum) and visualised with Alexa Fluor 488 (1:1000; A11001/A11005, Life Technologies Ltd) and 594 (1:1000; A11007, Life Technologies Ltd) conjugated IgGs. 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma Aldrich) staining was used to visualise the nuclei.
For fluorescence studies, eyes were collected at specified time points and fixed overnight in 4% paraformaldehyde at 4 °C. Post-fixation eyes were cryoprotected by incubation in 30% sucrose in PBS. Eyes were then frozen and cyrosectioned as previously described (22 (link)). Cryosectioned eyes were stained with mouse anti-Rho-1D4 (1:1500), rabbit anti-calnexin (1:600; C4731, Sigma Aldrich) and rat anti-prominin-1 (1:200; clone 13A4, CD133, eBioscience) primary antibodies in blocking solution (3% BSA, 10% normal goat serum) and visualised with Alexa Fluor 488 (1:1000; A11001/A11005, Life Technologies Ltd) and 594 (1:1000; A11007, Life Technologies Ltd) conjugated IgGs. 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma Aldrich) staining was used to visualise the nuclei.
3
Comprehensive Antibody Purchasing and Generation
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Monoclonal mouse Rab5 antibody (#50523; Abcam, Tokyo, Japan), rabbit polyclonal GFP antibody (GFP-Rb-Af2020; Frontier Institute, Ishikari, Hokkaido, Japan), mouse monoclonal FLAG antibody (F1804; Sigma-Aldrich), goat polyclonal EEA1 antibody (sc-6415; Santa Cruz Biotechnology, Dallas, TX, USA), goat polyclonal GST antibody (27–4577; GE Healthcare, Wauwatosa, WI, USA), Alexa Fluor 488-conjugated donkey anti-rabbit IgG antiserum, and Alexa Fluor 555-conjugated goat anti-mouse IgG antiserum (Invitrogen, Carlsbad, CA, USA) were purchased for immunohistochemistry and/or Western blot analysis. Polyclonal rabbit and guinea pig anti-podocin sera have been described previously20 (link).
Antibodies against calnexin (C4731; Sigma Aldrich, St. Louis, MO, USA), caveolin (ab2910; Abcam, Cambridge, UK), and LAMP1 (MAB4800; R&D Systems, Minneapolis, MN, USA) were purchased for subcellular fractionation. Antibody against the β subunit of mitochondrial F1F0-ATPase was prepared as described previously43 (link).
To generate antibodies against human SNX9, rabbits were immunized with a hemocyanin-conjugated peptide (single letter code, CFGHPQAYQGPATGDD) corresponding to the amino acids of human SNX9, and resulting antibodies were affinity-purified as described previously44 (link) (seeSupplementary Fig. S1 ).
Antibodies against calnexin (C4731; Sigma Aldrich, St. Louis, MO, USA), caveolin (ab2910; Abcam, Cambridge, UK), and LAMP1 (MAB4800; R&D Systems, Minneapolis, MN, USA) were purchased for subcellular fractionation. Antibody against the β subunit of mitochondrial F1F0-ATPase was prepared as described previously43 (link).
To generate antibodies against human SNX9, rabbits were immunized with a hemocyanin-conjugated peptide (single letter code, CFGHPQAYQGPATGDD) corresponding to the amino acids of human SNX9, and resulting antibodies were affinity-purified as described previously44 (link) (see
4
Antibody Dilutions for Western Blot and Immunostaining
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For western blots, the antibody against pan-syntrophin (MAI-745, Affinity BioReagents, Golden, CO, United States) was used at a dilution of 1/200. A custom rabbit polyclonal antibody against α1-syntrophin (Pineda Antibody Service, Berlin) was used at a dilution of 1/1000. The antibodies against Cre (69050; Novagen, EMD Millipore-MERK, Schaffhausen, Switzerland) was used at a dilution of 1/500 and the antibody against calnexin (C4731; Sigma-Aldrich, St. Louis, MO, United States) was used at 1/1000. For Duolink® experiments, the antibody against syntrophin (pan-syntrophin, MAI-745, Affinity BioReagents, Golden, CO, United States) was used at a dilution of 1/100 and custom rabbit polyclonal antibody against Nav1.5 (Pineda Antibody Service, Berlin) was used at a dilution of 1/200. For staining, the anti-pan-syntrophin antibody (MAI-745, Affinity BioReagents, Golden, CO, United States) was used at a dilution of 1/200. The antibody against Cre (69050; Novagen, EMD Millipore-MERK, Schaffhausen, Switzerland) was used at a dilution of 1/1000.
5
Quantification of Dynamin-2 in Muscle Samples
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For western blot, protein extracts from muscle samples were loaded on a 10% SDS PAGE, and membranes were incubated with rabbit anti-dynamin 2 (R2865; 1:700; Massana Munoz et al., 2019 (link)) and rabbit anti-calnexin (C-4731; Sigma-Aldrich; 1:1000), as well as with peroxidase-coupled goat anti-rabbit antibodies (111-035-144; Jackson ImmunoResearch; 1:10,000). The immunoblots were revealed with the SuperSignal West Pico kit (Thermo Fisher Scientific), and monitored on an Amersham Imager 600 (GE Healthcare Life Sciences). Quantification of band intensity was performed using the Measurement Log plugin of Adobe Photoshop 2022, version 23.1.0. The DNM2/calnexin ratio of the integrated grey level density was calculated for each sample and expressed as the percentage of the average ratio in control dogs. The individual ratio values were plotted using Prism 9 for MacOS, version 9.3.1.
6
Western Blot Analysis of MCU in Pancreatic Samples
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Pancreatic samples were homogenized on ice in RIPA buffer. The protein concentration of lysed samples was determined using Pierce BCA protein assay kit (Thermo Fisher Scientific). BisTris NuPAGE gels (4–12%; Invitrogen) were used for electrophoresis (30 μg of protein/lane) with MES SDS NuPAGE running buffer (Invitrogen). SeeBlue Plus2 molecular weight standards (Invitrogen) were employed. Proteins were transferred to Hybond-ECL nitrocellulose membrane (Amersham Biosciences). Blots were blocked for 1 h at room temperature in 5% milk dissolved in PBS with 0.05% Tween-20, pH 7.4. Primary antibodies used: HPA016480 (anti-MCU, rabbit poly-clonal, Sigma) 1:400 dilution; C4731 (anti-calnexin, rabbit poly-clonal, Sigma) 1:2000; and A6154 (HRP-conjugated goat anti-rabbit polyclonal, Sigma) 1:1000. The chemiluminescence signal was detected using Pierce Supersignal West Pico chemiluminescent reagents (Thermo Fisher Scientific) with a ChemiDoc XRS + molecular imaging system (Bio-Rad, Hercules, CA, USA).
7
Immunoblotting of Microsomal Proteins
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8
RyR1 and Calnexin Protein Analysis
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Microsomal proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane. Western blotting was performed using antibodies for RyR1 (34C, Developmental Studies Hybridoma Bank, University of Iowa, IA, USA) and calnexin (C4731, Sigma-Aldrich, MO, USA).
9
Hippocampal Cultures Protein Analysis
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14DIV hippocampal cultures were lysed in lysis buffer with 5% SDS and analyzed by Western blotting using mouse anti‐phospho ERK1/2 (1:1,000, M8159, Sigma‐Aldrich, Milan, Italy), rabbit anti‐calnexin (1:2,000, c4731, Sigma‐Aldrich, Milan, Italy), rabbit anti‐SHANK2 (1:2,000; 162202, Synaptic Systems, Goettingen, Germany), mouse anti‐PSD95 (1:10,000; UC Davis/NIH NeuroMab Facility, CA), rabbit anti‐SV2a (1:2,000; 119002, Synaptic Systems, Goettingen, Germany), guinea pig anti‐vGlut1 (1:1,000; 135304, Synaptic Systems, Goettingen, Germany), mouse anti‐SNAP‐25 (1:100,000; SMI81 Sternberger Monoclonals, Baltimore MD), rabbit anti‐GAPDH (1:4,000, 247002, Synaptic Systems, Goettingen, Germany). Membranes were washed and incubated for 1 h at room temperature with the secondary antibody anti‐mouse or anti‐rabbit HRP conjugated (Thermo Fisher, Waltham, MA, USA). Blots were scanned using a ChemiDoc‐MP system (Bio‐Rad, Segrate, Italy). Optical densities were measured using the Fiji software (NIH, Bethesda, Maryland, USA) with local background subtracted as described Fossati et al (2015 ). For each sample, calnexin was used as a loading control.
10
Western Blot and Immunostaining Protocols
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For western blots, a mouse monoclonal anti-dystrophin antibody (NCL-DYS2, Novocastra, Muttenz, Switzerland) was used at a dilution of 1/250. A mouse monoclonal antibody against SAP97 (VAM-PS005F, Enzo life science, Lausen, Switzerland) was used at a dilution of 1/500. Against Nav1.5, a custom-made rabbit polyclonal antibody (Pineda Antibody Service, Berlin) was used at a dilution of 1/200. A rabbit polyclonal antibody against Cre (69050; Novagen, EMD Millipore-MERK, Schaffhausen, Switzerland) was used at a dilution of 1/500 and a rabbit polyclonal antibody against calnexin (C4731; Sigma-Aldrich, Saint-Louis Missouri, USA) was used at 1/100011 (link),26 (link),27 (link).
For immunostainings, rabbit polyclonal antibody against Cre (69050; Novagen, EMD Millipore-MERK, Schaffhausen, Switzerland) was used at a dilution of 1/500 and mouse monoclonal anti-dystrophin (Dys NCL-DYS2, Novocastra, Muttenz, Switzerland) was used at a dilution of 1/25011 (link),26 (link),27 (link).
For immunostainings, rabbit polyclonal antibody against Cre (69050; Novagen, EMD Millipore-MERK, Schaffhausen, Switzerland) was used at a dilution of 1/500 and mouse monoclonal anti-dystrophin (Dys NCL-DYS2, Novocastra, Muttenz, Switzerland) was used at a dilution of 1/25011 (link),26 (link),27 (link).
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