Muscle Protein Analysis Post-Injury

Snap frozen whole TA muscles were crushed into powder using a mortar and pestle. Crushed muscles were then used for protein analysis at a sample size of n = 4 per group for the 56-day post-injury time point and n = 5 per group for the 28-day time point. Powdered muscle samples were suspended in T-PER™ (ThermoFisher; Waltham, MA, USA) lysis buffer and Halt™ Protease Inhibitor Cocktail 100× (Thermo Fisher Scientific Inc.; Waltham, MA, USA) at a concentration of 100 mg/mL. The suspended samples were homogenized and centrifuged at 10,000× g for 5 min at 4 °C. The supernatant was collected, and total protein concentration was quantified using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific Inc.; Waltham, MA, USA) according to the manufacturer’s instructions. Proteins were measured in muscle lysate using ELISA kits (MyBiosource Inc; San Diego, CA, USA). Proteins associated with fibrogenic processes included collagen type I alpha I (COL1a1), collagen 3 alpha I (COL3A1), and transforming growth factor beta (TGFβ). Proteins associated with myogenic processes included myogenin (MYOG), paired box 7 (PAX7), insulin-like growth factor (IGF-1), and myosin heavy chain 3 (MHC3). Protein lysates were further diluted in the T-PER™ lysis buffer as needed and added to antibody-coated plates along with standard protein concentrations. The plates were incubated and washed according to specific manufacturer instructions. A change in color appeared in the plate, and the optical density was measured at specific wavelengths using an Infinite M200 Pro spectrophotometer (Tecan; Männedorf, Switzerland). Fibrotic and myogenic protein concentrations were calculated by interpolation from the generated standard curve and normalized to the total protein concentration quantified from the BCA assay.