Methyl pp2a c

50 mg gastrocnemius muscle was dissected and homogenized in Buffer A (1% Triton X-100, 20 mmol Tris (pH 75), 2.5 mmol sodium pyrophosphate, 150 mM NaCl, 1 mmol EGTA, 1 mmol sodium vanadate, 2 mM beta-glycerophosphate, 1 μg/ml leupeptin, 1 ul/ml aprotinin, 1 ul/ml PMSF) using a PRO 200 homogenizer (PRO Scientific, Oxford, CT, USA). Samples were centrifuged (12000 rpm, 10 min at 4 °C) and protein concentrations of the supernatant determined by the Bio-Rad protein assay kit (Bio-Rad laboratories, Inc. Hercules, CA). Supernatants (80 μg) were resolved by SDS-PAGE and subjected to standard immunoblotting. Protein abundance was detected with antibodies against, Akt1, Akt2, phospho-Akt (ser 473), phospho-Akt (Thr 308), AMPKα1, AMPKα2, phospho AMPK (Thr 172) (Millipore, Temecula, CA). Other antibodies were PP2A-A, PP2A-B, PP2A-C (Cat No. SC-80665), methyl PP2A-C (Cat No. SC-81603) and phospho PP2A Tyr 307 (Cat No. SC-12615) all from Santa Cruz Biotechnology, Santa Cruz, CA). Protein expression levels were normalized by tubulin (Santa Cruz Biotechnology, Santa Cruz, CA). Phosphorylation or methylation levels were normalized by the corresponding protein expression. Optical densities of protein bands were analyzed using Image J. Data are expressed as the fold difference of the LFD control group.