Muscle tissue lysates were prepared by dissection and homogenized in buffer A (25 mmol/l HEPES, pH 7.4, 1% Nonidet P-40, 137 mmol/l NaCl, 1 mmol/l phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 1 μg/ml pepstatin, and 5 μg/ml leupeptin), using a PRO 200 homogenizer (PRO Scientific, Oxford, CT). The samples were centrifuged at 14,000g for 20 min at 4°C, and protein content of the supernatant was determined (Bio-Rad protein assay kit; Bio-Rad Laboratories, Hercules, CA). Supernatants (50 μg) were resolved by SDS-PAGE and subjected to immunoblotting using chemiluminescence reagent (PerkinElmer Life Science, Boston, MA) and quantified as described (22 (link)). The 19S proteasome base anti-S5A/Rpn10 antibodies were ordered from Calbiochem (Gibbstown, NJ). Antibodies for phospho–insulin receptor substrate (IRS)-1 (Tyr612), phospho–insulin receptor (IR) (Tyr1150/1151), phosphoinositol (PI) 3-kinase protein 85 (p85 of PI 3-kinase), phospho-Akt (Ser473), IRS-1 and IRS-2, 20S proteasome subunit β2i, Akt, serum- and glucocorticoid-inducible kinase 1 (SGK1), signal transducer and activator of transcription 3 (STAT3), and SIRT1 antibodies were obtained from Upstate Biotech (Lake Placid, NY). Anti-19S proteasome lid subunits S9/Rpn6 and S14/Rpn12 antibodies were ordered from BIOMOL International (Plymouth Meeting, PA). GLUT4 monoclonal antibody was obtained from R&D Systems (Minneapolis, MN). Lipoprotein lipase (LPL) antibody was purchased from GeneTex (San Antonio, TX) and β-actin from Affinity Bioreagents (Golden, CO). IR β-subunit was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).