Ponceau s red

Whole cell lysates were prepared using a RIPA buffer (Sigma-Aldrich), sonicated, and cleared of cellular debris by centrifugation at 14,000g for 15 min. TA muscles were snap-frozen in liquid nitrogen and homogenised with RIPA buffer (Sigma-Aldrich) using a DUALL Tissue Homogeniser (Kimble Kontes). Tissue was centrifuged at 3,000g for 10 min, and then 14,000g for 15 min prior to measuring concentrations of protein in the lysates using the Bradford Protein Assay (Thermo Fisher Scientific, Waltham, MA, United States). Western blot analyses were performed as described previously (Gao et al., 2008 (link)) using nitrocellulose membranes (Biorad; Hercules, CA, United States) and commercial antibodies to Myosin (Merck, Darmstadt, Germany). Equal protein loading and electroblot transfer were verified by staining the membrane post transfer with Ponceau S Red (Sigma-Aldrich). This method has been routinely used (Perry et al., 2018 (link)) and validated as a reliable measure of protein loading with immunoblotting (Romero-Calvo et al., 2010 (link)). Following application of Chemiluminescent Substrate (SuperSignal^TM West Pico PLUS, Thermo Fisher), densities of detected protein bands were recorded with a BioRad chemiluminescence imager (ChemiDoc XRS+, BioRad) and densities determined using Image J (NIH, Bethesda, MD, United States).

Cell lysates were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 12.5% polyacrylamide gels and transferred to nitrocellulose membrane in Tris-Glycine-ethanol buffer (25 mM Tris Base, 192 mM Glycine, 20% Ethanol) for 90 minutes at 400mAmps. Transfer of proteins was confirmed with staining with Ponceau S Red (Sigma). Nonspecific binding sites were blocked with 4% skim milk or BSA in PBS. Blots were incubated overnight at 4 °C in primary antibody diluted in 1% skim milk or BSA in PBST (PBS with 0.1% Tween 20). After overnight incubation blots were washed in PBST and incubated in species specific secondary antibody conjugated to horseradish peroxidase diluted 1:5000 in 1% skim milk in PBST. Bound antibodies were detected with Enhanced Chemiluminiscence (ECL, Perkin Elmer) and exposure on Li-Cor Odyssey Fc Imager. Where appropriate, digital images were analysed using ImageJ to estimate protein levels relative to tubulin and values expressed as arbitrary units normalised to tubulin.

Cells were lysed in ice-cold Lysis Buffer 6 (R&D systems) supplemented with 10 μL/mL of protease inhibitor cocktail. Total protein content was determined by Precision Red Reagent (Sigma) protein assay. Twenty micrograms of total protein were separated on 10% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore Co., Bedford, MA). Protein transfer was checked by staining the membrane with Ponceau S red (Sigma-Aldrich). The membrane was then blocked using 5% bovine serum albumin (BSA) (Sigma) or 5% milk (Sigma) in PBS supplemented with 0.05% Tween-20 (Sigma) (PBST). Primary antibodies RB1 (BD; Cat# 554136) and β-Actin (Abcam, Cat# ab8226) were incubated overnight at 4 C. After four washes with PBST, the membrane was incubated with a secondary antibody for 1 h at room temperature. Protein bands were detected using SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific) using the ChemiDoc XRS + System (Bio-Rad).