Rabbit monoclonal anti cleaved caspase 3 antibody

Apoptotic events were determined by Annexin V (BD Pharmingen™, #556547), cleaved caspase-3 (BD Pharmingen™, #560901) and terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) staining. For flow cytometry, cells were harvested and stained with both Annexin V and PI for 10 min. They were then washed by PBS, and resuspended in HEPES. For cleaved caspase-3 staining, deparaffinized sections were subjected to antigen retrieval in 0.01 mol/ml citrate buffer (pH 6.0) by microwave heating. After blocking with 5% Bovine Serum Albumin (BSA, Life Technologies Inc., Carlsbad, CA, USA), the sections were incubated with rabbit monoclonal anti–cleaved caspase-3 antibody (Cell Signaling Technologies Inc., Danvers, MA, USA). TUNEL assay was performed following the manufacturer's instructions. The nuclei were counterstained using DAPI. The number of positively stained nuclei was counted from 10 fields of view and 5 different groups.

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).

4T1 cells were cultured in T25 cell flasks for 24 h at 37 °C in 5% CO₂. Then, the cells were treated with DTX, Blank NPs, DTX/CA-Oxi-αCD NPs, or DTX/FA-CA-Oxi-αCD NPs at an equivalent concentration of 20 ng/mL DTX for 48 h, and the control was treated with cell culture medium. After 48 h, the cells were lysed and centrifuged, and the supernatant concentration was detected with a BCA kit (Beyotime Biotechnology Co., Ltd., Shanghai, China). The expression of cleaved-caspase-3 and phosphorylated Bcl-2 were analyzed by Western blot assay. In addition, the expression of the FA receptor on the surface of the 4T1 and MDA-MB-231 cells was also determined by Western blot analysis. The following antibodies were used for immunoblotting: rabbit monoclonal anti-cleaved-caspase-3 antibody (1:1000 dilution, Cell Signaling Technology, Inc. MA, USA), rabbit monoclonal anti-p-Bcl-2 antibody (1:1000 dilution, Cell Signaling Technology, Inc., Danvers, MA, USA), and rabbit monoclonal GAPDH antibody (1:1000 dilution, Cell Signaling Technology, Inc., Danvers, MA, USA), and FRα antibody (1:500, DF4058, Affinity Biosciences, OH, NJ, USA).

The cells were seeded onto two-well chamber slides and transfected for 72 h with TFF3-siRNA and siCtrl. After blocking with 1% BSA in TBS, the cells were incubated overnight at 4 ℃ with primary antibodies, including a rabbit polyclonal anti-TFF3 antibody (Santa Cruz), a rabbit monoclonal anti-cleaved caspase-3 antibody (Cell Signaling Technology), a mouse monoclonal anti-cytochrome c antibody (Santa Cruz) and a goat polyclonal anti-Smac antibody (Santa Cruz). The slides were rinsed with TBS and then incubated for 1 h with anti-rabbit IgG-FITC (Sigma), anti-mouse IgG-FITC (Santa Cruz) and anti-goat IgG-FITC (Santa Cruz). Then, the cell nuclei were stained with Hoechst 33342 (Life Technologies, Carlsbad, CA), and the mitochondria were stained with the Mito-ID Red detection kit (Enzo Life Sciences, Farmingdale, NY). The slides were analyzed by fluorescence microscope (Axio Imager M1, Carl Zeiss, Oberkochen, Germany). Three fields were randomly selected from three independent experiments.

The lung tissues and cells were harvested at the end of the experiment. Protein was extracted from tissue or cells in a lysis buffer. After estimate protein concentrations with a bicinchoninic acid protein assay kit, equal amounts of protein (40 μg) from each sample were separated and electrotransferred onto a PVDF membranes. The membranes were then blocked in 5% BSA and incubated overnight at 4 °C with primary antibodies. After washing three times in TBST, the membranes were then incubated with HRP-coupled secondary antibodies. The antibodies are as follows: rabbit monoclonal anti-cleaved caspase-3 antibody (1:1000, Cell Signaling Technology, USA), rabbit monoclonal anti-Bcl-2 antibody (1:1000, Cell Signaling Technology, USA), rabbit monoclonal anti-Bax antibody (1:1000, Cell Signaling Technology, USA), rabbit monoclonal anti-phospho-Akt antibody (1:1000, Ser473, Cell Signaling Technology, USA), anti-rabbit IgG, HRP- linked antibody (1:2500, Cell Signaling Technology, USA), rabbit polyclonal anti-β-actin antibody (1:2000, Solarbio, China). Bands were detected using standard ECL (Millipore, USA) and quantified by densitometric analysis using Image J software (Version 1.44p, National Institutes of Health, Bethesda, MD, USA).

The human ESCC cell line TE5, TE8, TE19m and TE15 was obtained from the Cell Resource Centre for Biomedical Research at the Institute of Development, Aging, and Cancer (Tohoku University, Sendai, Japan). The human ESCC cell lineLYSE150 and KYSE170 was obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). The cells were cultivated using our previously reported protocols²⁰ . 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. A rabbit monoclonal anti-TRPV2 antibody was used for the immunohistochemical analysis, and a protein assay was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The following antibodies were used in the Western blotting analysis: a rabbit monoclonal anti-caspase 3 antibody and rabbit monoclonal anti-cleaved caspase 3 antibody, which were purchased from Cell Signaling Technology (Beverly, MA). A mouse monoclonal anti-β-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA).