Procaspase 3

Protein levels in cells were measured using Western blot assay. The transfected cells were lysed in Triton lysis buffer (Solarbio, Beijing, P.R. China) for 30 min on ice, and the protein concentration was determined by Bicinchoninic Acid (BCA) Kit (Solarbio). The protein was resolved over 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (20 (link)). The membrane was blocked in blocking buffer (5% nonfat milk) for 2 h at room temperature, and then incubated with primary antibodies against p-Akt (1:1,000), Akt (1:1,000), Bcl-2 (1:1,000), Bax (1:1,000), p27 (1:1,000), p21 (1:1,000), procaspase 3 (1:1,000), cleaved caspase 3 (1:1,000), and actin (1:2,000) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. A secondary antibody (1:2,000) (Cell Signaling Technology, Danvers, MA, USA) was used for 1 h at room temperature. The bands were detected by chemiluminescence and autoradiography using an X-ray film (Applygen, Beijing, P.R. China), and densitometric measurements were performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Two of the gastric cancer cell lines, SNU-1, SNU-16 cells were obtained from the Laboratory of Cell Biology at the Cancer Research Institute in Seoul National University College of Medicine. They were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (Gibco BRL, Carlsbad, CA, USA), 100 U of penicillin and 100 μg/mL of streptomycin at 37°C in the humidified atmosphere of 95% air and 5% CO₂ in an incubator. Molecular mass markers for proteins were obtained from Pharmacia Biotech (Saclay, France). Antibodies against phospho-Akt (Ser473), Akt 1/2/3, X-linked inhibitor of apoptosis protein (XIAP), and procaspase 3 were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Antibodies against phospho-Akt (Thr 308), and phospho-p53 (Ser 15) were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Antibody against poly (adenosine diphosphate-ribose) polymerase (PARP) was purchased from BD Biosciences Pharmingen (San Diego, CA, USA). Antibody against β-actin was from Sigma (Beverly, MA, USA). Peroxidase-labeled donkey anti-rabbit and sheep anti-mouse immunoglobulin, and an enhanced chemiluminescence (ECL) kit were purchased from Amersham (Arlington Heights, IL, USA). All other chemicals not specifically cited here were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

Dithiothreitol solution, Rhodamine PE, 3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide (MTT), Annexin V-FITC apoptosis detection kit, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) were purchased from Sigma-Aldrich (St Louis, MO, USA). Anti-human KDR monoclonal antibody, VEGFR2/KDR, was from R&D System (Minneapolis, MN, USA). Egg yolk phosphatidylcholine was purchased from Northern Lipids Inc. (Burnaby, BC, Canada). Cholesterol was obtained from Shanghai Chemical Reagent Co. Ltd., (Shanghai, People’s Republic of China). Sodium morrhuate was from Shanghai Donghai Pharmaceutical Company (Shanghai, People’s Republic of China). RPM1640 medium, TRIzol reagent, and reverse transcription polymerase chain reaction (RT-PCR) reagent kit were obtained from Invitrogen (Grand Island, NY, USA). Anti-VIII antibody, biotinylated IgG, pro-caspase-3, pro-caspase-8, pro-caspase-9, Bcl-2, Bax, and β-actin were from Santa Cruz Biotechnology Co., Ltd (Dallas, TX, USA).