Anti p akt

Chemicals and drugs of the purest available grade including NAC were provided by Sigma Chemical Co. (St. Louis, MO, USA). Primary antibodies anti-P-AKT (reacting with Ser473), anti-AKT, anti-Insulin-receptor (anti-IR), anti-fructokinase (anti-fructokinase), anti-glutathione-peroxidase (anti-GPx), and anti-glutathione reductase (anti-GR) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA; catalog number 6040S, 9272,sc-711, sc-50029, sc-133160, and sc-133245, respectively). Anti-COX-2 from CAYMAN Laboratories (Ann Arbor, MI, USA catalog number 160106), anti-iNOS, and anti-eNOS were obtained from Sigma (St. Louis, MO, USA; catalog number N7782). Anti-P-eNOS (Ser 1177) was provided by Cell Signaling Laboratory (Danvers, MA, USA; catalog number N3893); anti-glucokinase antibody (sheep anti-GST-glucokinase fusion protein antibody) was kindly provided by Dr. Mark Magnusson (Vanderbilt University, TN, USA). This antibody, another from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA; glucokinase-N-19: sc:1980), and anti-GAPDH from Millipore (Carlsbad, CA, USA; catalog number 92590) were also provided. Finally, a secondary antibody anti-rabbit IgG Peroxidase (developed in goats) was obtained from Sigma (St. Louis, MO, USA; catalog number A9169).

Western blotting was performed as previous reported [36 (link), 37 (link)]. Briefly, Hep-2 cells were grown in the mid-log phase, washed with phosphate buffered saline and lysed for 30 min on ice. The lysis buffer with pH 6.8 contained 10% glycerol, 5% 2-mercaptoethanol, 2% sodium dodecyl sulfate (SDS), 62.5 mmol/L Tris-HCl. The SDS–PAGE analysis was performed and membranes were incubated overnight at 4°C with antibodies directed against the indicated proteins. The antibodies were obtained from the following companies: anti-TrkB, anti-β-actin, anti-p-Akt, anti-cyclin D1, anti-p-s-Src and anti-c-Src were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-RUNX3 and anti-Keap1 were purchased from Cell Signaling Technology (Danvers, MA, USA); anti-E-cadherin, anti-N-cadherin, anti-vimentin, anti-Twist-1 and anti-Twist-2 were purchased from BD Biosciences (San Jose, CA, USA). Membranes were washed, incubated with the appropriate secondary antibodies and exposed with the ECL chemiluminescent substrate kit (Pierce, Rockford, IL, USA). Images were analyzed with ImagePro (Media Cybernetics, Bethesda, MD, USA) and densitometry data were analyzed by using either conventional Student’s t-test. Results are reported as mean ± SD. A P-value <0.05 was considered significant and all were two-tailed.

Detection of pAKT, pHER2, caspase-9 activation and PARP cleavage was performed by Western blot according to standard procedures [18 (link)]. The primary antibodies were as follows: anti-caspase-9, anti-PARP and anti-pAKT were from Santa Cruz Biotechnology (Santa Cruz, CA), anti-pHER2 from Cell Signaling (Danvers, MA), and anti-β-actin from Sigma (St. Louis, MO).