P akt ser473

Protein expression was determined by Western blot analysis as previously described [7 (link)]. Briefly, 40 µg of protein lysate was denatured and loaded onto a 10% SDS-polyacrylamide gel and transferred to a polyvinylidene fluoride membrane. Nonspecific binding on the membranes was blocked using 5% w/v low-fat milk in Tris-buffered saline with Tween-20. Subsequently, the membrane was incubated overnight at 4 °C with the relevant Cell Signaling (Beverly, MA, USA) primary antibodies: Phosphorylated Protein kinase B at Ser 473 (p-AKTSer473; 1:1000), phosphorylated 5’ AMP-activated protein kinase at threonine 174 (p-AMPKThr174; 1:800), phosphorylated nuclear factor-kappa β phosphorylation at serine 536 (p-NF-kBSer536, 1:1000), phosphorylated Insulin receptor substrate-1 phosphorylation at serine 307 (p-IRS-1Ser307, 1:1000), protein kinase C phosphorylation at serine 660 (p-PKCSer660, 1:1000) and GLUT4 (1:800) (Sigma-Aldrich, Saint Louis, MO, USA). The relevant horseradish peroxidase-conjugated secondary antibodies were applied the following day for 90 min at room temperature. β-Actin (1:2000) (Santa Cruz Biotechnology, Dallas, TX, USA) antibody was added as a loading control. Proteins were detected and quantified using a Chemidoc-XRS imager and Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).