Pt308 akt

We used Proteo-JETTM Mammalian Cell Lysis Reagent (Thermo) to lyse cells. Crude lysates were centrifuged at 14,000g for 20 min at 4 °C. Lysate proteins were mixed with loading buffer and denatured by heating at 100 °C for 10 min. Proteins were resolved by SDS-PAGE and transferred to a PVDF membrane (Millipore, Billerica, MA, USA), blocked with 5% nonfat milk, and then incubated with primary antibodies overnight at 4 °C on a shaking table. Primary antibodies targeting E-cadherin (1:1000), Vimentin (1:1000), AKT (1:1000), AKT-pS473 (1:1000), AKT-pT308 (1:1000), ERK (1:1000), p-ERK (1:1000) (Cell Signaling Technology, Danvers, MA, USA), α-SMA (1:1000), TBK1 (1:1000) (Abcam, Cambridge, MA, USA), and GAPDH (Beyotime, China) were used. Then, the blots were incubated with the secondary antibody, HRP-conjugated IgG (Beyotime, dilution 1:5000), for 1 h at room temperature. Specific bands for each protein were detected with an Image Quant LAS4000 imager (GE Healthcare Life Science, Pittsburgh, PA) using Amersham ECL (Millipore). Densitometric analysis of expression was performed using ImageJ software (National Institutes of Health, Bethesda, MD). The expression of each molecule was normalized to GAPDH expression.

Tissues or cells were homogenized in a lysis buffer containing 100 mM Tris (Merck, Burlington, Massachusetts) pH7.5, 2 mM EDTA (Sigma-Aldrich, St. Louis, Missouri), 2 mM EGTA (Sigma-Aldrich, St. Louis, Missouri), 150 mM NaCl (Sigma-Aldrich, St. Louis, Missouri), 1% Triton X-100 (Fluka Chemie, Buchs, Switzerland), cOmplete inhibitor cocktail (Sigma-Aldrich, St. Louis, Missouri) and PhosSTOP (Sigma-Aldrich, St. Louis, Missouri). Protein concentration was determined by the Bradford assay (Bio-rad), and equal amounts of protein were separated by SDS-PAGE, and transferred onto nitrocellulose membranes (GE Healthcare, Chicago, Illinois). Antibodies used in this study were as follows: AKT (Cat#4685 or Cat#2920), AKT-pS473 (Cat#4060), AKT-pT308 (Cat#13038), PRAS40-pT246 (Cat#2997), PRAS40 (Cat#2691), HK2 (Cat#2867), S6-pS240/244 (Cat#5364), S6 (Cat#2217), FASN (Cat#3189), ACC (Cat#3662), HA tag (Cat#3724) and ChREBP (Cat#58069) from Cell Signaling Technology (Danvers, Massachusetts), GLUT4 (Cat# NBP2-22214, Bio-Techne, Abingdon, United Kingdom), HSP90, (Cat#sc-13119, Santa Cruz Biotechnology, Dallas, Texas), CALNEXIN (Cat#ADI-SPA-860-F, Enzo Life Sciences, Farmingdale, New York) and ACTIN (Cat#MAB1501, Merck, Burlington, Massachusetts). For quantification, the specific signals were normalized to a loading control.

Antibodies were purchased from Cell Signaling Technologies (USA): β-Tubulin (number 2128), rpS6-P(S235/236) (number 2211), 4E-BP1-nonP(T46) (number 4923), 4E-BP1 (number 9644), Akt-P(S473) (number 4060), Akt-P(T308) (number 4056), Akt (number 4685), Paxillin-P(T118) (number 69363), Paxillin (number 12065), and Actin (number 4970). Monoclonal mouse anti-human MVP (LRP-56) antibodies were purchased from Enzo (cat. no. ALX-801-005-C050, Enzo).