Pakt phospho ser473

Total protein extracts were separated in 5%–20% gradient acrylamide gel for SDS-PAGE, transferred to a nitrocellulose membrane and probed with specific antibodies against phospho-Akt (pAkt; phospho-Ser473, Cell Signaling, 1:1,000, Cat no. 4051, raised against synthetic phosphopeptide corresponding to residues around Ser473 of mouse Akt), Akt (40D4, Cell Signaling, 1:2,000, Cat no. 2920, detects the C-terminus of the protein), InsR (4B8, Cell Signaling, 1:1,000, Cat no. 3025, raised against synthetic peptide corresponding to residues surrounding Tyr999 of human insulin receptor β), IGF-1R (Cell Signaling, 1:1,000, Cat no. 3027, detects the C-terminus of the protein) and mouse anti-β-actin (Sigma-Aldrich Cat no. A5441), at 4°C for 12 h. The membranes were initially blocked with 5% nonfat milk or BSA in TBS-T (0.25 M Tris-Base, 8% NaCl, pH 7.6, 0.1% Tween 20). The membranes were washed four times for 10 min in TBS-T and incubated with secondary anti-mouse IgG-HRP (Dianova, 1:20,000, Cat no.111-035-146) or anti-rabbit IgG-HRP (Dianova, 1:20,000, Cat no. 111-035-144) antibodies for 1 h 30 min. The membranes were washed again and developed with an ECL system (Luminata Western HRP Substrate from Millipore or SuperSignal West Pico from Thermo Fisher Scientific, Waltham, MA, USA).