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.

Antibodies against CIP2A (lot number K2420), PP2Ac (lot number D2309), or β-actin (lot number J0421) and horseradish peroxidase (HRP)-conjugated secondary antibodies were from Santa Cruz Biotechnology (Dallas, Texas, USA). Antibodies against PSA (Lot number 3), PLK1 (lot number 6), phospho-c-Myc (Ser62) (lot number 5), or c-Myc (lot number 15) were purchased from Cell Signaling (Danvers, MA, USA). Antibodies against phospho-AR (Ser81) (lot number 3497306) or AR (lot number 3256650) were obtained from Millipore (Burlington, MA, USA). Enzalutamide was obtained from MedChemExpress (Monmouth Junction, NJ, USA). LB100 and BI6727 were purchased from BioVision (Milpitas, CA, USA). Okadaic acid, TD52, Bicalutamide, and other chemicals were obtained from Sigma-Aldrich (Burlington, MA, USA).

Ishikawa cells were treated with EBC buffer (Roche Diagnostics) and 1 mM phosphatase inhibitor mixture II (Sigma-Aldrich). SDS-PAGE was applied by transferring the proteins onto nitrocellulose membranes. 5% nonfat milk in PBS with Tween 20 was subsequently utilized to block the membranes. Afterward, the membranes were incubated with primary antibodies against GAPDH (CST, Cat. No. 5174S, 1 : 1000), FBXL 16 (Merck, Cat. No. SAB2103554, 1 : 1000), PP2A-^Aα/β (Santacruz, Cat. No. sc-13600, 1 : 800), PP2A^B55a (Santacruz, Cat. No. sc-81606, 1 : 800), PP2A^C (Santacruz, Cat. No. sc-13601, 1 : 800), cyclin D1 (Abcam, Cat. No. ab182858, 1 : 3000), cyclin D1 phosphor T286 (Abcam, Cat. No. ab62151, 1 : 1000), AKT1 phosphor T308 (Abcam, Cat. No. ab278565, 1 : 1000), GSK3β (Abcam, Cat. No. ab32391, 1 : 2000), and ki-67 (Abcam, Cat. No. ab184787, 1 : 1000) overnight at 4°C. After being washed with 1× TBST, the membranes were incubated with respective secondary antibodies conjugated with horseradish peroxidase for 1 h at room temperature. The protein bands were visualized with Immobilon™ Western Chemiluminescent HRP Substrate (Millipore Corporation, Cat. No. WBKLS0500), and the images were captured on the visualization instrument Tanon-5200 (Tanon, China).