Anti phospho akt ser473 paktser473

Unless otherwise noted, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO) or Fisher Scientific. Sodium dodecyl sulfate-polyacrylamide electrophoresis apparatus, immunoblotting reagents were obtained from Bio-Rad Laboratories (Hercules, CA). Pierce MemCode Reversible Protein Stain Kit, BCA Protein Assay Kit and Pierce Detergent Compatible Bradford Assay Kit were from Thermo Fisher (Waltham, MA). Anti-phospho-AKT Ser473 (pAKTSer473; #9271), anti-phospho-AKT Thr308 (pAktThr308; #9275), anti-phospho-AKT2 Ser474 (pAKT2Ser474; #8599), anti-phospho-AS160 Thr642 (pAS160Thr642; #8881), anti-AKT1 (# 2938), anti-AKT2 (#3063), anti-Na⁺, K⁺ ATPase (#3010), anti-LDH (#3558), anti-insulin receptor (IR; #3025) and anti-rabbit IgG horseradish peroxidase conjugate (#7074) were from Cell Signaling Technology (Danvers, MA). Anti-AKT substrate of 160 kDa (AS160; ABS54) was obtained from EMD Millipore. Human recombinant insulin was from Eli Lilly (Indianapolis, IN).

The protein levels PTEN, PI3K, AKT, mTOR, FOXO3a and FOXO3a phosphorylated (pFOXO3a) in the TA muscle were analyzed using Western blotting. Samples were loaded to SDS-PAGE on polyacrylamide gels (6–15%). After electrophoresis, proteins were electro-transferred to a nitrocellulose membrane (BioRad Biosciences, Hercules, CA, USA). Equal loading of samples (30μg) and transfer efficiency were monitored with the use of 0.5% Ponceau S staining of the blot membrane. The blot membranes were incubated with polyclonal antibodies anti-AKT (#9272, Cell Signaling Tech, Danvers, MA, USA), anti-phospho-AKT-SER473 ([pAKT-SER473] #9271s, Cell Signaling Tech, Danvers, MA, USA), anti-PI3K (#ab32569, Abcam, Cambridge, UK), anti-PTEN (#9559, Cell Signaling Tech, Danvers, MA, USA), anti-mTOR (#2972, Cell Signaling Tech, Danvers, MA, USA), FOXO3a (#ab12162, Abcam, Cambridge, UK), FOXO3ap (#ab15478, Abcam, Cambridge, UK) and anti-GAPDH (ab37168, Abcam, Cambridge, UK). The bands were analyzed using Image J software (Image J Corporation based on NIH image, Bethesda, MD, USA). Skeletal muscle GAPDH expression levels were used to normalize the results, which are expressed as a percentage of control expression.