β tubulin

Total protein lysates were obtained from EDL- and SOL-derived myoblasts and myotubes. After exposure to RIPA buffer (Thermo Fisher Scientific, Yokohama, Japan). Primary antibodies (Piezo1, Proteintech, 15939-1-AP) and β-tubulin (Wako, 10G10) diluted 1:100 and 1:5000 respectively in 5% skim milk, were incubated either for 90 min (β-tubulin) or at 4 °C overnight (Piezo1). Samples were then incubated with HRP-conjugated secondary antibodies (Cell signalling, anti-rabbit, 7074P2, and anti-mouse, 7076S) in 5% skimmed milk at 1:5000 dilution for 1 h at room temperature. Samples were visualised by chemiluminescence with a digital luminescent image analyser LAS-4000 (GE Healthcare, Tokyo, Japan).

The following antibodies were used: mtIF2 (1:200, Santa Cruz Biotechnology, Dalla, TX, USA, sc‐365477); mtIF3 (1:2000, Origene, Rockville, MD, USA, TA800421); mtEFTu (1:5000, Abcam, Cambridge, UK, ab173300); mtEFTs (1:10,000, Abcam, ab173528); mtEFG1(1:3000, Abcam, ab173529); mtRF1L (1:1000, Proteintech, Rosemont, IL, 16694‐1‐AP); mtRRF1 (1:1000, Proteintech, 12357‐2‐AP); mtEFG2 (1:1000, Proteintech, 16941‐1‐AP); TACO1 (1:3000, Proteintech, 21147‐1‐AP); insulin receptor substrate 1 (IRS‐1, 1:3000, Cell Signaling Technology, Danvers, MA, USA, #3194); hormone sensitive lipase (HSL, 1:3000, Cell Signaling Technology, #18381); glyceraldehyde 3‐phosphate dehydrogenase (GAPDH, 1:10000, Wako, Osaka, Japan, 016‐25523); β‐tubulin (1:10,000, Wako, 014‐25041).
We used the Total OXPHOS Rodent WB Antibody Cocktail (1:3000, Abcam, ab110413) to detect the proteins involved in mitochondrial oxidative phosphorylation (OXPHOS). This cocktail contains five monoclonal antibodies: mitochondrial NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 8 (NDUFB8, ab110242); mitochondrial succinate dehydrogenase [ubiquinone] iron‐sulfur subunit (SDHB, ab14714); mitochondrial cytochrome b‐c1 complex subunit 2 (UQCRC2, ab14745); mitochondrial encoded cytochrome c oxidase I (MTCO1, ab14705); mitochondrial ATP synthase subunit alpha (ATP5A, ab14748).

Antibodies specific to GPER1 (ab39742; Abcam, Cambridge, MA, USA), β-actin (sc-47778; Santa Cruz Biotechnology, Dallas, TX, USA), α-tubulin (017-25031; FUJIFILM Wako Pure Chemical Corporation), and β-tubulin (014-25044; FUJIFILM Wako Pure Chemical Corporation) were used. Whole-cell extracts were prepared, and Western blot analysis was performed as previously described [32 (link)]. The protein concentrations of whole-cell extracts were determined using the RC DC^TM Protein Assay (Bio-Rad, Hercules, CA, USA). The polyvinylidene difluoride membrane was stained with Ponceau S solution (Beacle Inc., Kyoto, Japan). Pink-stained blots were scanned, and the images were converted to grayscale for publication. Band intensities were quantified using the ImageJ 1.46r software “https://imagej.nih.gov/ij/ (accessed on 21 September 2022)”. The values obtained for the GPER1 band were normalized to the intensity of the β-actin band (internal control).