Caspase 9 antibody

Protein lysis buffer (Intron Biotechnology, Seoul, South Korea) was used for the protein extraction from A2780 cells. Protein concentrations were measured by the Bradford assay. The protein samples were mixed with SDS-PAGE sample buffer and heated for 5 min at 95 °C. The samples were loaded on a gel for SDS-PAGE. Following electrophoretic separation, separated proteins were blotted to PVDF (polyvinylidene difluoride) membranes. After blocking with 2% bovine serum albumin (BSA) for 30 min, the membranes were incubated overnight at 4 °C with diluted primary antibodies against caspase-3, -8, 9, and β-actin in TBS-T (Tris-buffered saline containing Tween-20) with 2% BSA. After a subsequent washing with TBS-T, the membranes were incubated with an appropriate secondary antibody at room temperature for 2 h. Immunoreactive bands were visualized by the ECL kit and analyzed by ImageQuant Las-4000 (GE Healthcare Life Science, WI, USA). Caspase-3 and β-actin antibodies were acquired from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Caspase-8 antibody was obtained from BD Biosciences. Caspase-9 antibody was purchased from Cell Signaling (Beverly, MA, USA).

The cells were treated with (−)-asarinin (1) and incubated for 48 h. The cells were rinsed twice with ice-cold PBS and lysed with protein lysis buffer (Intron Biotechnology, Seoul, Korea) containing protease inhibitors (0.5 mM PMSF and 5 μg/mL aprotinin). The lysates were mixed with 5X sodium dodecyl sulfate (SDS) sample buffer and boiled for 5 min for denaturation. Total protein was run on 10–12% SDS-PAGE gels and electrotransferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was immunoblotted using specific primary antibodies overnight at 4 °C following blocking with 5% non-fat dry milk for 30 min–1 h. After washing, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (1:1000–2000) at room temperature for 1−2 h. After washing, immunepositive bands were visualized using an ECL chemiluminescent system and analyzed by Image Quant LAS-4000 (Fujifilm Life science, Tokyo, Japan). Anti-caspase-3 and b-actin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Caspase-9 antibody was purchased from Cell Signaling (Beverly, MA, USA). Caspase-8 antibody was obtained from BD Biosciences (San Jose, CA, USA).

Protein was isolated from BGC-823 and SGC-7901 cell lines as previously stated [19 (link)].
Forty micrograms of total protein s were run on a 14.7% polyacrylamide gel, and then transferred to polyvinylidene difluoride membranes (Hybondenhanced chemiluminescence; Amersham Pharmacia Biotech). The antibodies against FBWX7(1:1000) and β-actin (1:1000) were purchased from Abcam (Abcam, Cambridge, MA, USA). The antibodies against Caspase-9 antibody (1:1000) and Caspase-3 (1:1000) were purchased from Cell Signaling Technology (Cell Signaling Technology, USA). The protein was measured with a Phototope–horseradish peroxidase Western blot detection kit (Cell Signaling Technology, Inc.), and β-actin was treated as an internal control.

Phorbol 12-myristate 13-acetate (PMA), PI, caffeine, and DMSO were purchased from Sigma-Aldrich (St. Louis, MO, USA). Z-VAD-fmk, Ac-LEHD-cho, Ac-IETD-cho, and Ac-DNLD-cho were purchased from Peptide Institute, Inc. (Osaka, Japan). Cleaved caspase-3 antibody (#9661), caspase-8 antibody (#9746), caspase-9 antibody (#9502), PARP (46D11) rabbit monoclonal antibody (#9532), β-actin antibody (#4967), anti-rabbit IgG horseradish peroxidase (HRP)-linked antibody (#7074), anti-mouse IgG HRP-linked antibody (#7076), phosphor-ATM (Ser1981) (D25E5) antibody (#13050), and Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (#4412) were purchased from Cell Signaling Technology Japan, K.K. (Tokyo, Japan). The fluorescein isothiocyanate (FITC)-labeled monoclonal antibody anti-human cluster of differentiation 95 (CD95-FITC) was purchased from BioLegend (San Diego, CA, USA). FITC-conjugated mouse IgG₁ antibody was purchased from Beckman–Coulter (Fullerton, CA, USA).