Multi gage software

One microgram of protein was denatured in 5X SDS sample buffer, boiled for 10min and then loaded onto 10% Tris-HCl ready gels (Bio-Rad Laboratories, Hercules CA, USA). Protein was then transferred onto a nitrocellulose membrane (Bio-Rad Laboratories) by electrophoresis at 60v (constant) for 2 hours. The membrane was blocked with 5% nonfat dried milk in TBS that contained 0.1% tween-20 (TBS-T). Membrane was then incubated overnight with primary antibody against the phosphorylated form of Akt (S473) (Cell Signaling, Danvers, MA) diluted in 3%BSA/TBS-T at 4°C with constant shaking. Membrane was washed with TBS-T three times for 5min each and incubated with HRP-conjugated anti-rabbit secondary antibody for 1 hour at room temperature, followed by washing steps with TBS-T. Band intensity was developed using the SuperSignal® West Dura Extended Duration Substrate (Pierce Biotechnology Inc. Rockford, IL). The blots were then scanned using the Luminescent Image Analyzer LAS 4000 (FujiFilm Medical Systems USA, Inc., Stamford, CT, USA) and band intensity was determined using MultiGage software (FujiFilm Medical Systems USA).