E-Cadherin antibody was obtained from BD Biosciences. Vimentin antibody was obtained from Sigma. Antibodies to cyclophilin, GSTP1, β-actin, phospho-AMPK α (Thr172), AMPK α, phospho-ACC (Ser79), ACC, phospho-S6, total S6, phospho-JNK (Thr183/Tyr185), JNK, and GAPDH were obtained from Cell Signaling Technology. FLAG antibody was obtained from Cayman Chemicals.
Cells were lysed in lysis buffer (CST) containing both protease and phosphatase inhibitors. Proteins were resolved by electrophoresis on 4–15% Tris-glycine precast Mini-PROTEAN TGX gel (BioRad Laboratories) and transferred to nitrocellulose membranes using the iBlot system (Invitrogen). Blots were blocked with 5% nonfat milk in Tris-buffered saline containing Tween-20 (TBST) solution for 1 hour at room temperature, washed in TBST, and probed with primary antibody diluted in recommended diluent per manufacturer overnight at 4°C. Following washes wit h TBST, the blots were incubated in the dark with a IR-linked secondary antibody at room temperature for 1 hour. Blots were visualized using an Odyssey Li-Cor scanner after additional washes.
Cells were lysed in lysis buffer (CST) containing both protease and phosphatase inhibitors. Proteins were resolved by electrophoresis on 4–15% Tris-glycine precast Mini-PROTEAN TGX gel (BioRad Laboratories) and transferred to nitrocellulose membranes using the iBlot system (Invitrogen). Blots were blocked with 5% nonfat milk in Tris-buffered saline containing Tween-20 (TBST) solution for 1 hour at room temperature, washed in TBST, and probed with primary antibody diluted in recommended diluent per manufacturer overnight at 4°C. Following washes wit h TBST, the blots were incubated in the dark with a IR-linked secondary antibody at room temperature for 1 hour. Blots were visualized using an Odyssey Li-Cor scanner after additional washes.