Glyceraldehyde 3 phosphate dehydrogenase gapdh

7,8-DHF was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich). Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), bovine calf serum (BCS), penicillin-streptomycin, and 2-NBDG were purchased from Invitrogen (Carlsbad, CA, USA). The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, Inner Salt (MTS) assay kit was obtained from Promega (Madison, WI, USA). The enzyme-linked immunosorbent assay (ELISA) kits for TNF-α, IL-6, MCP-1, and adiponectin were obtained from R&D Systems (Minneapolis, MN, USA). Nuclear and cytoplasmic extraction reagents and the assay kit for NEFA were provided by Thermo Fisher Scientific (Rockford, IL, USA) and FUJIFILM Wako Pure Chemical Corporation (Chuo-Ku, Osaka, Japan), respectively. Antibodies specific for phospho-c-Jun N-terminal kinases (p-JNK), total-JNK (t-JNK), phospho-Akt (Ser473), Akt, and NF-κB were purchased from Cell Signaling Technology (Danvers, MA, USA). The antibody for lamin B was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and antibodies for α-tubulin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were from Ab Frontier (Seoul, Korea). All other chemicals were obtained from Sigma-Aldrich.

Cells were washed with phosphate-buffered saline (PBS) and then lysed with cell lysis buffer (Cell Signaling Technology), as described by the manufacturer. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). After blocking with blocking buffer composed of Tris-buffered saline (TBS) containing 0.1% Tween 20 and 5% skim milk (BD), for 1 h at room temperature, the membranes were incubated with the following antibodies: A-Raf, B-Raf, C-Raf, CagA, and Urease A (1:1,000, Santa Cruz Biotechnology), EGFR, phospho EGFR, Mek1/2, Erk1/2, and phospho Erk1/2 (1:1,000, Cell Signaling Technology), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1,000, AbFrontier) overnight at 4°C. Subsequently, membranes were washed three times with TBS containing 0.1% Tween 20 and then incubated with horseradish peroxidase-conjugated Goat anti-rabbit IgG (Santa Cruz Biotechnology) or Goat anti-mouse IgG (Santa Cruz Biotechnology) for 1 h at room temperature. The protein bands were visualized using the enhanced chemiluminescence solution (Advansta) as described by the manufacturer.