Enhanced chemiluminescence ecl kit

Anthocyanins isolated from the fruit of V. coignetiae Pulliat were a generous gift from Dr S.C. Shin (Department of Chemistry, Gyeongsang National University, Jinju, Korea) (22 (link)), and 100 mg/mL concentration stock solution was made by dissolving in distilled water. Dulbecco’s modified Eagle’s medium (DMEM), bovine calf serum (BCS), and other tissue culture reagents were obtained from WelGENE Inc (Daegu, Korea). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), differentiation medium (MDI), and TG assay kit were purchased from Sigma-Aldrich Chemical Co (St. Louis, MO, USA). Bio-Rad protein assay kit was obtained from Bio-Rad Laboratory (Hercules, CA, USA). Primary antibodies (Table 1) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and Cell Signaling Technology, Inc (Danvers, MA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies and enhanced chemiluminescence (ECL) kit were purchased from Amersham Biosciences (Westborough, MA, USA). All other chemicals not specifically mentioned here were purchased from the Sigma-Aldrich Chemical Co.

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).

Human AGS cells from the American type culture collection (Rockville, MD, USA) were cultured in RPMI 1640 medium (Invitrogen Corp, Carlsbad, CA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) (GIBCO BRL, Grand Island, NY, USA), 1 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in a humidified atmosphere of 95% air and 5% CO₂. Antibodies against Bcl-2, Bid, Bcl-xL, phospho p53, p53, BAX, c-IAP-2, X-linked IAP (XIAP), Akt, phospho Akt, ERK, phospho ERK, and procaspase 3 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody against poly (ADP-ribose) polymerase (PARP) was purchased from 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). All other chemicals not specifically cited here were purchased from Sigma Chemical Co. (St. Louis, MO, USA).