Total akt

Pelleted cells were homogenized by sonication in lysis buffer [20 mM Tris-HCl, pH 7.4, 5 mM EDTA, 10 mM Na₄P₂O₇, 100 mM NaF, 1% Nonidet P-40, 2 mM Na₃VO₄, protease inhibitor (10 μL per mL) and phosphatase inhibitor (20 μg per mL)], followed by centrifugation at 1200 × g for 10 min at 4 °C. Protein concentrations in supernatants were determined using a bicinchoninic acid (BCA) protein assay kit (23255; Pierce, Thermo Fisher Scientific) and bovine serum albumin as standard. Cell lysates (20 µg protein) were subjected to standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis and proteins were transferred to a nitrocellulose membrane. Proteins were reversibly visualized using MemCode stain (24580; MemCode Reversible protein Stain, Pierce, Thermo Fisher Scientific) and detected using antibodies against phosphor-AKT^Ser473 (sc-33437, Santa Cruz Biotechnology), total AKT (05-591, Millipore), phospho-FOXO1^Thr24/FOXO3a^Thr32 (#9464, Cell Signaling), total FOXO1 (#2880, Cell Signaling), phospho-acetyl-CoA carboxylase^Ser79(#3661, Cell Signaling), and total acetyl-CoA carboxylase (#3661, Cell Signaling). Densitometric analysis was performed using Image lab 5.0 software from BioRad. Protein expression data were corrected to protein stain.

Cells were assayed as previously described [45 (link)]. Primary antibodies were as follows: total β-catenin was from BD Transduction Laboratories, total NHERF1 from ThermoFisher, total c-Myc, cyclin D1, p27, Rab7, RILP, V1G1, Cathepsin D, and α-Tubulin were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), total and phosphorylated pERK1/2 (pThr202/Tyr204) MAPK, total AKT, LC3, Beclin-1 (BECN1), and PARP were from Millipore.

HA and Ran antibodies were obtained from Abcam (ab18181, ab157213). Kapβ2 antibody was obtained from Santa Cruz Biotechnology (sc-11368). GAPDH and histone antibody were purchased from Cell signaling Technology (2118, 8135). PTEN, p-AKT and total AKT antibodies were purchased from Millipore, USA (04-035, 05-1003, 16-294).