Immediately following euthanasia, LD samples were stored at −70°F until processing. Free intracellular AA concentrations in muscle were determined by high-performance liquid chromatography (HPLC; PICO-TAG reverse-phase column; Waters, Mildford, MA,) using an analytical method based on deproteinization and derivatization of AA with phenylisothiocyanate, as previously described (18 (link)).
Amino acid transporter and intracellular markers of protein degradation and autophagy were measured by immunoblotting analysis, as previously described (8 (link), 31 (link)). The antibodies used in the immunoblotting process were PKB (total and Ser473, Cell Signaling Technology, Beverly, MA), AMPK-α (total and Thr172, Cell Signaling Technology, Beverly, MA), FOXO1 (total protein, Santa Cruz Technology, Santa Cruz, CA; Ser256, Cell Signaling Technology), FOXO4 (total protein and Ser262, Cell Signaling Technology, Beverly, MA), pro-caspase 3 (total protein, Cell Signaling Technology, Beverly, MA), MuRF1 (ECM Biosciences, Versailles, KY), atrogin-1 (ECM Biosciences, Versailles, KY), LAT1 (Cell Signaling Technology, Beverly, MA), SNAT2 (Aviva System Biology, San Diego, CA.), LC3 (Cell Signaling Technology, Beverly, MA), β-actin (Santa Cruz Biotechnology, Santa Cruz, CA), α-Actin (40 KDa; Dako-Cytomation, Glostrup, Denmark) and LAMP-2 (Cell signaling Technology, Danvers, MA)