Ppi3k and pi3k

Total protein was extracted using RIPA lysis buffer, and equal amounts of proteins were subjected to 8% SDS-PAGE and then transferred to polyvinylidene difluoride membranes (Millipore Corp., MA, U.S.A). The membranes were blocked and then incubated in primary antibodies against vimentin, β-catenin, AAT), insulin receptor substrate-1 (IRS-1) (1:1000, Cell Signaling Technology, Beverly, MA), pAkt and Akt (1:1000, Cell Signaling Technology), pPI3K and PI3K (1:1000, Cell Signaling Technology), and GAPDH (1:10,000 Sigma-Aldrich) overnight at 4 °C with gentle agitation, followed by incubation in the anti-rabbit IR-Dye 670- or 800cw-labeled secondary antibody for 1 h at room temperature. Two 10-min washes in TBST were performed after secondary antibody labeling; then, the membranes were placed in TBST. The membranes were imaged using a LiCor Odyssey scanner. Boxes were manually placed surrounding each band of interest to measure the raw near-infrared fluorescence intensity values, and the intra-lane background intensity was subtracted using Odyssey 3.0 analytical software (LiCor, Lincoln, NE, USA).

Monoclonal antibodies specific for Beclin‐1 and p62 were purchased from Sigma‐Aldrich. The anti‐GAPDH monoclonal antibody was acquired from Calbiochem‐Merck Chemicals Ltd. Similarly, anti‐active caspase‐3, anti‐SIRT‐1, p‐PI3K and PI3K, p‐mTOR and mTOR, and anti‐Parkin were obtained from Cell Signaling Technology. Finally, anti‐Bcl‐2, anti‐Bax, anti‐ATG12, and anti‐MAP‐LC3 antibodies were obtained from Santa Cruz Biotechnology. A cocktail of protease inhibitors (Complete™ Protease Inhibitor Cocktail) was purchased from Boehringer Mannheim. The Immun Star HRP substrate kit was obtained from Bio‐Rad Laboratories Inc.