Rabbit monoclonal anti caspase 3 antibody

RA-FLS and OA-FLS were cultured in DMEM medium containing 10 % FetalClone 1 serum (Thermo Scientific, USA), 100 U/ml penicillin, and 100 mg/ml streptomycin (Sigma-Aldrich, St Louis, MO) in a humidified incubator at 37 °C under 5 % CO₂. Apoptosis was induced in the cells by adding 15 μM staurosporine (Sigma-Aldrich, St Louis, MO) or 20 μM Smac 060 or Smac 066 for 6 h. FLS were then collected for Western blot analysis. To detect caspases, we used an anti-caspase 3 rabbit monoclonal antibody (Cell Signalling Technology) and an anti-caspase 8 antibody (Cell Signalling Technology); β-actin (Sigma-Aldrich, St Louis, MO) was used as the loading control.

Western blotting was performed by loading 30 μg protein on 10% (w/v) tris-glycine denaturing gels and separating the proteins by electrophoresis, then transferring them to a PVDF membrane. After blocking, the membrane was incubated with primary antibodies, anti-PARP rabbit monoclonal antibody (1:1000, Cell Signaling Technology, Invitrogen Fisher Scientific, Illkirch, France), anti-caspase-3 rabbit monoclonal antibody (1:1000, Cell Signaling Technology), anti-Bax rabbit monoclonal antibody (1:1000, Cell Signaling Technology), anti-Bcl-2 rabbit monoclonal antibody (1:1000, Cell Signaling Technology) at +4 °C overnight. Following incubation, the membrane was incubated with anti-β-actin mouse monoclonal antibody (1:1000, Cell Signaling Technology) at room temperature for 1 h. After washing, the membrane was incubated with infrared labeled secondary antibodies (Odyssey Western Blotting kit, LI-COR Biosciences, Lincoln, NE, USA) at room temperature for 1h. Proteins were visualized, and quantified by scanning the membrane on an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA) with both 700- and 800-nm channels.

Human ESCC cell lines TE5 (poorly differentiated type) and TE15 (well differentiated type) were obtained from the Cell Resource Center for Biomedical Research at the Institute of Development, Aging, and Cancer (Tohoku University, Sendai, Japan). Human ESCC cell line KYSE70 (poorly differentiated type) was obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). These cell lines were grown in RPMI-1640 medium (Nacalai Tesque, Kyoto, Japan) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% fetal bovine serum (FBS). Cells were cultured in flasks and dishes in a humidified incubator at 37°C in 5% CO₂ in air. The monoclonal anti-AQP1 antibody used for the immunohistochemical analysis, immunofluorescence analysis, and protein assay was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The following antibodies were used in the western blotting analysis; rabbit polyclonal anti-Jun-amino-terminal kinase (JNK) antibody, rabbit monoclonal anti-phosphoJNK antibody, rabbit monoclonal anti-Caspase 3 antibody, and rabbit monoclonal anti-Cleaved-Caspase 3 antibody were purchased from Cell Signaling Technology (Beverly, MA). A mouse monoclonal anti-β-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA).