Western blotting

A portion of muscle homogenates isolated during the myofibrillar protein extractions was used for western blotting analysis. Phosphorylation status and total protein content of mammalian target of rapamycin complex 1 at Ser2448 (mTORC1: 1° dilution 1:500, catalogue number 2971; Cell Signaling Technology, Beverly, MA USA), 70 kDa S6 protein kinase 1 at Thr389 (p70S6K: 1° dilution 1:500, catalogue number 9205; Cell Signaling Technology), eukaryotic initiation factor 4E binding protein 1 at Thr37/46 (4E‐BP1: 1° dilution 1:1000, catalogue number 9459; Cell Signaling Technology) and nuclear factor‐kappa B (NF‐κB) at Ser468 (NF‐κB: 1° dilution 1:1000, catalogue number 3039; Cell Signaling Technology) were determined using antibodies purchased from Cell Signaling Technology. Muscle protein content was also determined for Toll‐like receptor 4 (TLR4: 1° dilution 1:500, catalogue number 13556; Abcam, Cambridge, MA, USA) and myeloid differentiation factor 88 (MyD88: 1° dilution 1:1000, catalogue number 4283; Cell Signaling Technology). Protein content of the homogenate was determined by Bradford Assay (Bio‐Rad, Hercules, CA, USA) and then equal amounts of protein were separated by SDS‐PAGE before being transferred to either nitrocellulose (mTORC1 and TLR4) or polyvinyl difluoride membranes (p70S6K, 4E‐BP1, MyD88 and NF‐κB). After blocking with either 5% BSA (mTORC1, 4E‐BP1, MyD88, NF‐κB) or 5% milk (p70S6K, TLR4), membranes were incubated in primary antibodies overnight at 4°C. Membranes from the respective proteins were then incubated with appropriate secondary antibodies and protein content was detected using West Femto Maximum Sensitivity substrate (SuperSignal; Thermo Scientific, Waltham, MA, USA) and the ChemiDoc XRS+ Imaging System (Bio‐Rad) and by comparing molecular weights with pre‐stained protein standards (Kaleidoscope; Bio‐Rad). After detection of phosphorylated proteins, membranes were stripped with western blot stripping buffer (Restore; Thermo Scientific) and re‐incubated with antibodies against total protein. Western blot data were normalized to an internal control (α‐tubulin: 1° dilution 1:5000, catalogue number 4074; Abcam). Bands were quantified using ImageJ (NIH, Bethesda, MD, USA), normalized to a control sample run on each blot to account for inter‐blot variability.