Blue xb 1

Western Blot analysis was performed as described previously [10 (link)]. Supernatants of cell homogenates containing equivalent amounts of protein were subjected to SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA). The membranes were incubated for 2 h at room temperature with primary anti-PEPCK antibody that attaches to both cytosolic and mitochondrial PEPCK) (Catalog No. sc-32879; Santa Cruz Biotechnology, Inc., Dallas, TX), VDAC (Abcam, Cambridge, UK), actin (Capital Bioscience, Rockville, MD), Bax (Abcam), Bcl-2 (Abcam), Bad (Abcam), AKT (Cell Signaling Technology, Inc., Danvers, MA), 4EBP1 (Cell Signaling Technology, Inc.), mTOR (Cell Signaling Technology, Inc.), p-AKT (Ser 473; Cell Signaling Technology, Inc.), p-mTOR (Ser 2448; Cell Signaling Technology, Inc.), or p-4EBP1 (Thr 70; Cell Signaling Technology, Inc.). The membranes were washed, incubated with diluted HRP-conjugated secondary antibody (SouthernBiotech, Birmingham, AL), and exposed to film (Blue XB-1; Kodak, Rochester, NY).

Western blot analysis was performed as described previously [30 (link)]. Briefly, protein samples were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA) and blocked in 5% non-fat dry milk for 1 h at room temperature. Membranes were incubated with primary antibody against calgranulin B (Santa Cruz Biotechnology, Dallas, TX, USA), aurora A kinase (Abcam, Cambridge, MA, USA), c-Caspase3 (Cell signaling, Massachusetts, USA), p-AKT (Cell signaling), p-ERK (Cell signaling), p-JNK (Cell signaling), NF-κB (Cell signaling), p53 (Cell signaling), p38 (ABcam), β-catenin (ABcam), E-cadherin (Cell signaling), and β-actin (Santa Cruz Biotechnology) or actin (Sigma-Aldrich, St. Louis, MO, USA). Membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Southern Biotech, Birmingham, AL, USA). Finally, membranes were rewashed (3 × 15 min), incubated with WEST-ZOL^® chemiluminescence reagent (iNtRON Biotechnology, Seoul, Korea) for 1 min and exposed to film (Blue XB-1, Kodak, Rochester, NY, USA).