Immunoblotting.

Protein levels of proteolytic (MURF1, Atrogin-1, 20 s proteasome subunit C8 and total ubiquitinated proteins), apoptotic (Bax, Bcl-2, and caspase-3), and autophagy (p62 and LC3B) markers, as well as total protein levels and phosphorylated ratios of nuclear factor κB (NF-κB) pathway markers (NF-κB p50 subunit, p-NF-κB p50/NF-κB p50, NF-κB p65 subunit, and p-NF-κB p65/NF-κB p65), MAPK pathway markers (p38 MAPK and p-p38 MAPK/p38 MAPK), and Forkhead box O (FOXO) pathway markers (FOXO3 and p-FOXO3/FOXO3) were analyzed using immunoblotting procedures, as previously described (26–28). Briefly, frozen samples from all experimental groups were homogenized in lysis buffer: 50 mM HEPES, 150 mM NaCl, 100 mM NaF, 10 mM Na pyrophosphate, 5 mM EDTA, 10% glycerol, 0.5% Triton-X, 5 µg/mL aprotinin, 2 µg/mL leupeptin, 100 µg/mL PMSF and 10 µg/mL pepstatin A. Afterward, samples were centrifuged at 3,600 rpm for 30 min and protein levels were determined in the supernatant using the Bradford method (Bio-Rad). Thirty micrograms of total protein were loaded on gels, then proteins were separated by electrophoresis, transferred to polyvinylidene difluoride (PVDF) membranes, blocked with bovine serum albumin (BSA), and incubated overnight with the corresponding primary antibodies: MURF1 (anti-MURF1, Santa Cruz Biotechnology, Dallas), Atrogin-1 (anti- MAFbx, Santa Cruz Biotechnology), 20 s proteasome subunit C8 (anti-C8 antibody, Biomol, Plymouth Meeting, PA), total ubiquitinated proteins (anti-ubiquitin, Santa Cruz Biotechnology), Bax (anti-Bax, Santa Cruz Biotechnology), Bcl-2 (anti-Bcl-2, Santa Cruz Biotechnology), caspase-3 (anti-caspase-3, Santa Cruz Biotechnology), p62 (anti-p62/SQSTM1, Merck KGaA, Darmstadt, Germany), LC3B (anti-LC3B, Cell signaling technology, Danvers, MA), NF-κB p50 (anti- NF-κB p50, Santa Cruz Biotechnology), p-NF-κB p50 (anti- p-NF-κB p50, Santa Cruz Biotechnology), NF-κB p65 (anti- NF-κB p65, Santa Cruz Biotechnology), p-NF-κB p65 (anti-p-NF-κB p65, Santa Cruz Biotechnology), p38 (anti-p38 α/β, Santa Cruz Biotechnology), p-p38 [anti-p-p38 (Tyr 182) Santa Cruz Biotechnology], FOXO3 (anti-FoxO3, Origene Technologies, Rockville, MD), p-FOXO3 (anti-p-FoxO3, Origene Technologies), and the endogenous control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (anti-GAPDH antibody, Santa Cruz Biotechnology).

Antigens from all samples were detected with horseradish peroxidase (HRP)-conjugated secondary antibodies and a chemiluminescence kit (Pierce ECL Western Blotting Substrate, ThermoFisher Scientific, Waltham, MA). For each of the antigens, samples from the different groups were always detected in the same picture under identical exposure times. PVDF membranes were scanned with the Alliance Q9 Advanced Chemiluminescence Imager (UVITEC, Cambridge, UK). The optical densities of specific proteins were quantified using the software Alliance Q9 (UVITEC). The mean values of the optical densities obtained in each specific group were calculated. To validate equal protein loading among various lanes, the glycolytic enzyme GAPDH was used as the protein loading controls in all the immunoblots.