Quantitative Analysis of SR-Related Proteins

SR‐related proteins were analyzed by Western blotting as previously described (Eshima, Tamura, et al., 2017 (link)). The protein abundance of the ryanodine receptor (RyR), the dihydropyridine (DHPR), the calsequestrin (CSQ), and the SR Ca²⁺‐ATPase (SERCA) was assessed. Briefly, polyvinylidene fluoride membranes were incubated overnight at 4°C with the following primary antibodies: anti‐type 1 ryanodine receptor (RyR) antibody 34C (MA3‐925; Thermo Fisher Scientific); anti‐dihydropyridine (DHPR) antibody 20A (ab2864; Abcam); anti‐calsequestrin antibody VIIID12 (MA3‐913; Thermo Scientific); anti‐SR Ca²⁺‐ATPase 2 (SERCA2) antibody 2A7‐A1 (MA3‐919; Thermo Scientific), and anti‐ glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) antibody 14C10 (no. 2118; Cell Signaling Technology) at 4°C. The membranes were incubated with the appropriate secondary antibody conjugated to horseradish peroxidase, enhanced by SuperSignal West Dura and Femto extended duration substrate (Thermo Fisher Scientific), and quantified by densitometry (C‐DiGit, LI‐COR Biosciences).

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Yamamoto H., Eshima H., Kakehi S., Kawamori R., Watada H, & Tamura Y. (2022). Impaired fatigue resistance, sarcoplasmic reticulum function, and mitochondrial activity in soleus muscle of db/db mice. Physiological Reports, 10(18), e15478.

Publication 2022

MA3‐925 MA3‐919 GAPDH) antibody 14C10 SuperSignal West Dura Femto extended duration substrate C‐DiGit

Anti antibody Antibodies Antibody Antibody a1 Ca2 atpase Calsequestrin Densitometry Digit Dihydropyridine Dura Glyceraldehyde 3 phosphate dehydrogenase Horseradish peroxidase Membranes Polyvinylidene fluoride Proteins Ryanodine receptor Serca2 Type 1 ryanodine receptor

Corresponding Organization : Juntendo University

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Protein abundance of the ryanodine receptor (RyR)
Protein abundance of the dihydropyridine (DHPR)
Protein abundance of the calsequestrin (CSQ)
Protein abundance of the SR Ca2+-ATPase (SERCA)

control variables

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used as a loading control

controls

Positive control: None mentioned
Negative control: None mentioned

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