Foxo3a ser253

The gastrocnemius muscle was homogenized in an extraction buffer containing protease and phosphatase inhibitors (Roche Diagnostics GmbH, Sandhoferstrasse, Mannheim, Germany). After centrifugation, the supernatant was subjected to protein quantification determined by the Bradford assay (Bio-Rad, Hercules, CA, USA) using an albumin standard curve. Samples were diluted in Laemmli’s buffer and submitted to SDS polyacrylamide gel electrophoresis (Sodium dodecyl sulfate (SDS)-PAGE) (25 (link)), transferred to a Polyvinylidene fluoride (PVDF) membrane, and incubated with primary antibodies against Akt (ref. 4685), Akt Ser473 (ref. 4058), total FoxO3a (ref. 9467), FoxO3a Ser253 (ref. 9466) (Cell Signaling Technology Danvers, MA, USA), or GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA, ref. SC 25778). They were then incubated with a peroxidase-conjugated anti-IgG antibody and after incubated with the peroxidase substrate (ECL Clarity TM, Bio-Rad, Hercules, CA, USA). GAPDH expression was used as an internal control. Images were obtained using the Amersham Imager 600 equipment (GE Healthcare, Buckinghamshire, UK) and quantified by optical densitometry using Image J software (National Institute of Health, Bethesda, MD, USA).