Srebp2

Cell lysates were prepared by washing cells twice with cold PBS and lysing cells in RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS, 1 mM EDTA, protease inhibitors) on ice for 30 min. Lysates were cleared by centrifugation and protein concentrations were determined using the Pierce 660 nm Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein were diluted in Laemmli sample buffer, boiled for 5 min and resolved by SDS-polyacrylamide gel electrophoresis. The resolved proteins were then transferred onto nitrocellulose membranes. Membranes were then blocked for 1 h in 5% milk in tris-buffered saline/0.1 % Tween-20 (TBS-T) at room temperature, then probed with the following primary antibodies in 5% milk/TBS-T overnight at 4 °C: SREBP-2 (1:250, BD Biosciences, 557037), p44/42 MAPK (ERK1/2) (1:1000, Cell Signaling Technology, 4695), PARP (1:1000, Cell Signaling Technology, 9542 L), ɑ-Tubulin (1:3000, Calbiochem, CP06) and E-cadherin (1:1000, Cell Signaling Technology, 3195). Primary antibodies were detected using IRDye-conjugated secondary antibodies (1:20,000, LI-COR Biosciences, 926-32211 and 926-32210) and the Odyssey Classic Imaging System (LI-COR Biosciences). Densitometric analysis was performed using ImageJ v1.47 software.

Livers from mice and Huh-7 cells were lysed in RIPA buffer (30 mmol/L Tris, pH 7.5, 150 mmol/L sodium chloride, 1  mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L sodium orthovanadate, 1% Nonidet P-40, 10% glycerol, and phosphatase and protease inhibitors). Western blotting was performed on 30–50-μg protein samples using commercially available antibodies against the following antigens: EGFR, phospho-EGFR (pEGFR), Akt, phospho-Akt (pAkt), fatty acid synthase (FASN), anti-sterol responsive elementary binding protein 1 (SREBP1), and SREBP2 (all from BD Biosciences, San Jose, CA, USA). Secondary antibodies (goat anti-mouse and goat anti-rabbit) were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA).