Tissues were lysed with lysis buffer (Beyotime Inc, Shanghai, China) followed by collection of cell protein. Protein concentration was determined using a BCA (bicinchoninic acid) protein kit (Invitrogen-Life Technologies). Equal amounts of denatured protein were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and transferred into nitrocellulose membranes. These membranes were blocked in Tris-buffered saline containing 0.1 % Tween 20 for 1 hour. Then the membranes were incubated with primary antibodies against Bax, Bcl-2 (Santa Cruz Biotechnology, Inc, Santa Cruz, CA, USA), cytochrome c, cleaved caspase 3 and 9, cleaved PARP, or CD69 (Cell Signaling Technology, Inc, Danvers, MA, USA) overnight at 4°C with shaking. After the membranes were washed 3 times, horseradish peroxidase (HRP)-conjugated secondary antibodies (Beyotime Inc) were added for 1 hour. After ECL chemiluminescence staining, bands were scanned and quantified using a chemiluminescence imaging system (Bio-Rad).
Cleaved caspase 3 and 9
Manufactured by Cell Signaling Technology
Sourced in United States
Cleaved caspase-3 and -9 are laboratory reagents used to detect the activation of caspase-3 and caspase-9, two key enzymes involved in the apoptosis (programmed cell death) pathway. These reagents can be used in various cell-based and biochemical assays to monitor cellular responses to apoptotic stimuli.
Automatically generated - may contain errors
Lab products found in correlation
Bcl 2, by Cell Signaling Technology (3 mentions)
Cleaved parp, by Cell Signaling Technology (2 mentions)
P erk1 2, by Cell Signaling Technology (2 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (2 mentions)
P jnk, by Cell Signaling Technology (2 mentions)
P erk, by Cell Signaling Technology (2 mentions)
Chemiluminescence imaging system, by Bio-Rad (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Beyotime (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (1 mentions)
Bcl 2, by Santa Cruz Biotechnology (1 mentions)
Fluorescence tagged secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Cell culture materials, by Thermo Fisher Scientific (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Cytiva (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
Cyclin d, by Cell Signaling Technology (1 mentions)
Mmp 2, by Cell Signaling Technology (1 mentions)
8 protocols using cleaved caspase 3 and 9
1
Protein Expression Analysis by Western Blot
2
Immunostaining Phospho-H2AX and Caspase Activation
3
Western Blot Analysis of Apoptosis Markers
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The total protein extracts were submitted for expression of Bcl-2 (1:1000), cleaved caspase-3 and -9 (1:1000), Bax (1:1000), cleaved PARP (1:1000), iNOS (1:500), p53 (1:1000), Nrf2 (1:500), ERK1/2 (1:1000), p-ERK1/2 (1:1000), JNK (1:1000), p-JNK (1:1000), HO-1 (1:1000), p38 (1:1000) were bought from Cell signaling Tech. USA and were estimated. The bands were viewed on Invitrogen iBright Imaging Systems (ThermoFisher USA).
4
Antitumor Effects of Polyphyllin F
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The Second Affiliated Hospital of Soochow University Institutional Animal Care and Use Committee approved this study. U87 and U251 human glioma cell lines were obtained from the Chinese Academy of Medical Sciences (Beijing, China). Cells were cultured in Dulbeccos modified Eagles medium (DMEM; HyClone Laboratories, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) in a 5% CO2 atmosphere at 37°C. Primary antibodies against Skp2, P21, P27, p-AKT (S473), AKT, extracellular signal-regulated kinase (ERK), p-ERK, cyclin D, p-cyclin D, cleaved caspase-3 and −9, MMP2, MMP9 and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). Secondary antibodies were from Liankebio (Hangzhou, China). Lipofectamine 3000 was from Invitrogen (Carlsbad, CA, USA). PF was from Tianjin Shilan Technology Co., Ltd., (Tianjin, China) and had a purity of 98%. PF was diluted in DMEM at a stock concentration of 400 mM. Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Laboratories (Kumamoto, Japan).
5
Polyphyllin VII-Induced Apoptosis Mechanisms
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Polyphyllin VII (purity, 99%; Fig. 1A ) was obtained from Beijing Solarbio Science and Technology Co., Ltd. All cell culture-related reagents were purchased from Invitrogen (Thermo Fisher Scientific, Inc.). Propidium iodide (PI), DAPI dye and 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay were obtained from Sigma-Aldrich (Merck KGaA). In addition, N-acetyl-L cysteine (NAC) was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. The specific antibodies against Bcl-2, Bax, LC3-I/II, ERK, p-JNK, JNK, p62, p-ERK, cleaved caspase-3 and −9, cleaved poly (ADP-ribose) polymerase (PARP), p-p38, p38 and GAPDH were purchased from Cell Signaling Technology, Inc. Finally, the class III PI 3-kinase inhibitor (3-MA) was obtained from Tocris Bioscience.
6
Modulation of Notch Signaling in Cancer Therapy
7
Protein Extraction and Quantification in Brain Tissues
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The total proteins, nuclear proteins, and cytoplasmic proteins in brain tissues or PC12 cells were extracted and quantified using the BCA Protein Quantification Kit (Pulilai Gene Technology Co. Ltd., Beijing, China). The intensity of Bax, Bcl-2, cytochrome c, and cleaved caspase-3 and -9 (Cell Signaling Technology, MA, USA) was assessed from the cerebral cortex region. The intensity of NF-κB p65, p-NF-κB p65, and HMGB1 (Cell Signaling Technology, MA, USA) and the intensity of TLR4, TRAF6, and MyD88 (Abcam, Cambridge, UK) were assessed from both the cerebral cortex and hippocampus regions. The SDS buffer was then added to the protein and boiled for 10 min. The SDS-PAGE electrophoresis was carried out. Then, the proteins were transferred onto PVDF membranes and blocked with 5% BSA for 2 h at room temperature. Proteins were then reacted with different kinds of antibodies at 4°C overnight. After the incubation, the secondary antibodies were introduced and reacted for 1 h. The protein expressions of different samples were observed using the High Sensitivity Chemiluminescence Detection Kit (Kangwei Biological Company, Beijing, China).
8
Lentiviral-Mediated Nrf2 Regulation in APL
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Chemicals and materials. α-tubulin and Nrf2 antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Cleaved caspase-3 and -9, cdc2 and cyclin B antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). DCfH-DA, propodium iodide (PI) and trypan blue were procured from Sigma. All of the other chemicals used were of the highest grade available commercially.
Cell culture. The human APL NB4 and HL-60 cell lines were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (fBS), 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco, Grand Island, NY, USA) in a humidified incubator at 37˚C with 5% CO 2 .
Transfection of lentivirus and plasmids. The PBK/TOPK-RNAi-lentivirus was constructed by Shanghai GeneChem Co., Ltd. (Shanghai, China). NB4 and HL-60 cells were transduced with the PBK/TOPK-RNAi-lentivirus and purified by puromycin treatment. Transduction efficiency of the cells was examined for expression of PBK/TOPK by real-time PCR. In some experiments, cells were transfected with the plasmid vector or plasmid expressing Nrf2 (Shanghai GeneChem) using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions.
Cell culture. The human APL NB4 and HL-60 cell lines were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (fBS), 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco, Grand Island, NY, USA) in a humidified incubator at 37˚C with 5% CO 2 .
Transfection of lentivirus and plasmids. The PBK/TOPK-RNAi-lentivirus was constructed by Shanghai GeneChem Co., Ltd. (Shanghai, China). NB4 and HL-60 cells were transduced with the PBK/TOPK-RNAi-lentivirus and purified by puromycin treatment. Transduction efficiency of the cells was examined for expression of PBK/TOPK by real-time PCR. In some experiments, cells were transfected with the plasmid vector or plasmid expressing Nrf2 (Shanghai GeneChem) using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions.
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