Total eif4e

Identical amounts of protein from cultured HepG2 cells and frozen liver tissue homogenates were separated by SDS-PAGE, transferred to PVDF membranes, and immunoblotted with primary antibodies followed by incubation with an HRP-conjugated secondary antibody. Finally detection procedures were performed using Immobilon Western Chemiluminescent HRP Substrate (Millipore, Temecula, CA, USA). Primary antibodies against the following proteins were used: CD36, total p70S6K, p-p70S6K (Thr 421/Ser 424), total 4E-BP1, p-4E-BP1 (Ser 65/Thr 70), total eIF4E, p-eIF4E (Ser 209) and β-actin (Santa Cruz, Dallas, TX, USA); total mTOR (Millipore, Temecula, CA, USA); and p-mTOR (phospho S2448) (Abcam, Cambridge, UK).

Protein lysates were prepared using RIPA lysis buffer (Sigma-Aldrich, ST. Louis, MO) with the addition of the complete EDTA-Free Protease Inhibitor Cocktail (Roche, Mannheim, Germany) and the HALT Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA). Equivalent amounts of protein were loaded per lane, separated by electrophoresis using 4–12% Bis-Tris gels (BIORAD, Hercules, CA) and transferred onto PVDF membranes (Invitrogen, Carlsbad, CA). Membranes were probed with the following primary antibodies: pAKT S473 (Cell Signaling Technologies, Beverly, MA) (CST-4060), pPRAS40 T246 (CST-2997),pP70S6K T389 (CST-9205), pS6RB S235/236 (CST-4858), p4EBP1 S65 (CST-13443),c-MYC (CST-13987), pEIF4E S209 (CST-9741), Total EIF4E (CST-9742), Cleaved PARP (CST-9546), Beta-Actin (Santa-Cruz Biotechnology, Dallas, TX) (SC-47778). After secondary antibody incubations, proteins were detected on film with the ECL chemiluminescent detection reagent (GE Healthcare Life Sciences, Pittsburg). The beta actin blots corresponding to Fig 3A–3D can be found in S1 Fig and demonstrate that equal amounts of protein were loaded per lane.