The lower lobe of the left lung was homogenized in lysis buffer, and the proteins were collected. For the detection of Nrf2 and NF-κB, the cytoplasmic component and nuclear component were isolated by treating a nuclear protein extraction kit (Beyotime Biotechnology, Shanghai, China) and centrifuged at 12,000 g for 10 min at 4°C. After the protein concentration was measured by a BCA kit, 50-μg protein samples were separated by SDS-PAGE and transferred onto a PVDF membrane. The membrane was blocked in 5% non-fat dry milk, followed by incubation with pro-caspase-3 (1:8,000, Abcam, United States), cleaved-caspase-3 (1:500, Abcam, United States), pro-caspase-9 (1:800, Abcam, United States), cleaved-caspase-9 (1:1,000, Abcam, United States), Nrf2 (1:5,000, Abcam, United States), and NF-κB (1:3,000, Abcam, United States) primary antibodies overnight at 4°C. After being washed and incubated with secondary antibody (1:5,000, Zhongshan Golden Bridge Biotechnology, Beijing, China) at room temperature for 1 h, the proteins were visualized with the enhanced chemiluminescence reagent (GE Healthcare Bio-Sciences, Pittsburgh, PA, United States), analyzed using ImageJ version 1.61 software (National Institutes of Health, Bethesda, MD, United States) and normalized to β-actin and Lamin B.
Pro caspase 9
Manufactured by Abcam
Sourced in United States
Pro-caspase-9 is a protein that plays a critical role in the process of apoptosis, or programmed cell death. It is the inactive precursor of the enzyme caspase-9, which is a key initiator of the caspase cascade that leads to cell death. Pro-caspase-9 must undergo proteolytic cleavage to become active caspase-9.
Automatically generated - may contain errors
Lab products found in correlation
Pro caspase 3, by Abcam (6 mentions)
Cleaved caspase 3, by Abcam (4 mentions)
Cleaved caspase 9, by Abcam (4 mentions)
Bcl 2, by Abcam (3 mentions)
Bcl2 associated x (bax), by Abcam (3 mentions)
Active caspase 3, by Abcam (2 mentions)
β actin, by Abcam (2 mentions)
Enhanced chemiluminescence reagent, by GE Healthcare (1 mentions)
Nuclear protein extraction kit, by Beyotime (1 mentions)
Nf κb, by Beyotime (1 mentions)
Nf κb, by Abcam (1 mentions)
Anti procaspase 8, by Abcam (1 mentions)
P akt, by Bioworld Technology (1 mentions)
Enhanced chemi luminescence (ecl), by Applygen (1 mentions)
Mercuric chloride hgcl2, by Merck Group (1 mentions)
P perk, by Abcam (1 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
Active caspase 9, by Abcam (1 mentions)
Mitosox red mitochondrial superoxide indicator, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
7 protocols using pro caspase 9
1
Analyzing Apoptosis Signaling Pathways
2
Protein Expression Quantification by Western Blot
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Total protein was extracted and protein concentrations established via bicinchoninic acid (BCA) assay. Protein (25 μg) was denatured, separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. After blocking overnight at 4°C using 5% BSA, the membranes were incubated with primary antibodies (anti-procaspase-8 1:2,500 and procaspase-9 1:2,000; both from Abcam, Cambridge, MA, USA), p-Akt 1:800 (Bioworld, St. Louis Park, MN, USA) for 2 h at room temperature, washed by TBST and incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody at 1:2,000 dilution for 2 h. Bands were visualized using enhanced chemiluminescence (ECL; Applygen, Beijing, China) detection reagents and scanned images were quantified using ImageJ software. Experiments were performed in triplicate with β-actin used as a housekeeping control for normalization. The ratio of target gene to β-actin was used for semi-quantification and comparison between different groups.
3
Mercuric Chloride Induced ER Stress
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Mercuric chloride (HgCl2, purity > 99.5%; Sigma-Aldrich, St. Louis, MO) was dissolved in 0.1 mol sterile phosphate buffered saline (PBS ) and added to mediums to provide Hg concentrations of 0 (Control), 5, 10, and 15 μmol/L. 4-Phenyl butyric acid (4-PBA , ER stress inhibitor) was obtained from Sigma Chemical Co. (St. Louis, MO).
Antibodies of PERK, p-PERK, CHOP, ATF4, GRP78, Pro-caspase-3, Cleaved caspase-3, Pro-caspase-9, Cleaved caspase-9, Bcl-2, Bax, and glyceraldehyde-3-phosphate dehydrogenase were from Abcam (Cambridge, England). The CEK cells were purchased from the Cell Biology Institution (Otwo, Shenzhen, China). They were cultured in high-glucose Dulbecco's Modified Eagle Medium (DMEM, Gibco, Pittsburgh, PA) containing 15% fetal bovine serum (Hyclone, Logan, UT) and incubated in a humidified 5% CO2 and 95% air incubator (Thermo Fisher Scientific, Grand Island, Maldives) at 37°C. Four independent experiments were performed in a parallel manner (n = 4). Each experiment had 4 concentrations, and each concentration had 4 parallel wells.
Antibodies of PERK, p-PERK, CHOP, ATF4, GRP78, Pro-caspase-3, Cleaved caspase-3, Pro-caspase-9, Cleaved caspase-9, Bcl-2, Bax, and glyceraldehyde-3-phosphate dehydrogenase were from Abcam (Cambridge, England). The CEK cells were purchased from the Cell Biology Institution (Otwo, Shenzhen, China). They were cultured in high-glucose Dulbecco's Modified Eagle Medium (DMEM, Gibco, Pittsburgh, PA) containing 15% fetal bovine serum (Hyclone, Logan, UT) and incubated in a humidified 5% CO2 and 95% air incubator (Thermo Fisher Scientific, Grand Island, Maldives) at 37°C. Four independent experiments were performed in a parallel manner (n = 4). Each experiment had 4 concentrations, and each concentration had 4 parallel wells.
4
Antioxidant and Apoptosis Modulators
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Resveratrol, dimethylsulfoxide (DMSO), N-acetyl-L-cysteine (NAC), and methylthiazolyldiphenyl-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP nick end labeling (TUNEL) kit was purchased from Roche Inc. (Germany). MitoSOX™ Red mitochondrial superoxide indicator was purchased from Invitrogen (Molecular Probes, Invitrogen, OR, USA). 2′-7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) was purchased from Beyotime Institute of Biotechnology (Jiangsu, China). The antibodies against SOD2, CAT, SULT1A1, and SULT1C2 were purchased from Proteintech (Chicago, IL, USA), and pro-caspase-3, active-caspase-3, pro-caspase-9, and active-caspase-9 from Abcam (Cambridge, UK).
5
Protein Expression Analysis via Western Blot
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Total proteins were isolated by RIPA Lysis Buffer (Beyotime, Shanghai, China). Proteins’ concentrations were tested by bicinchoninic acid (BCA) kit (Beyotime). The protein sample was separated on SDS/PAGE gel, and transferred to polyvinylidene fluoride membranes, followed by the blockage with 5% nonfat milk for 1 h. Next, the membranes were probed with primary antibodies of ZDHHC5 (1:1000, Proteintech), Bcl-2 (1:1000, Abcam), Bax (1:1000, Abcam), pro-caspase-3 (1:1000, Abcam), cleaved-caspase-3 (1:1000, Abcam), pro-caspase-9 (1:1000, Abcam), cleaved-caspase-9 (1:1000, Abcam), COX-2 (1:1000, Abcam), and GAPDH (1:1000, Beyotime) overnight at 4°C. Then, membranes were incubated with secondary antibody (1:1000, Beyotime) for 2 h keeping in a dark place at room temperature. The protein levels were detected by enhanced chemiluminescence (ECL) Plus reagent (Beyotime).
6
Apoptosis Induction in Gastric Cancer
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F12, RPMI‐1640 medium and foetal bovine serum (FBS) were purchased from Gibco, BRL (Grand Island, NY, USA). PGE2 ELISA kit ADI‐900‐001 was purchased from Enzolifesciences (Farmingdale, NY, USA). Hoechst 33258 was purchased from Sigma‐Aldrich (St Louis, MO, USA). The Annexin V‐FITC Apoptosis Kit was purchased from Becton Dickinson (Franklin Lakes, NJ, USA). Mitochondrial Membrane Potential Assay Kit was purchased from Beyotime Biotechnology (Jiangsu, China). The primary antibodies against ATF‐4, Chop, Bax, Bcl‐2, Mcl‐1, Puma, Noxa, Bim, Bak, procaspase‐3, active caspase‐3, procaspase‐9, poly ADP‐ribose polymerase (PARP) and β‐actin were purchased from Abcam Inc. (Cambridge, MA, USA). Human gastric carcinoma cell line AGS and HGC‐27 cells were purchased from Shanghai Institutes for Biological Sciences, CAS (Shanghai, China). Cells were cultured with F12 or RPMI 1640 medium containing 10% FBS and 1% penicillin/streptomycin at 37°C, 5% CO2 humidified atmosphere. Both ABT‐737 and DMC were dissolved in Dimethyl sulfoxide (DMSO) at the concentration of 100 mM.
7
Protein Expression Analysis of AgNP-Treated Cells
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The total protein extraction kit with a protease inhibitor cocktail (Thermo Pierce Co., Ltd., Wilmington, DE, USA) and a BCA quantitation kit (Haogene Co., Ltd., Hangzhou, China) were used to determine the total protein and total protein quantitation of the RTE cells treated with AgNPs. The blots were incubated with cytoc (1: 1000), pro-cas-pase3 (1: 1000), cleaved-caspase3 (1: 200), pro-caspase9 (1: 1000), cleaved-caspase9 (1: 1000), and β-actin (1: 1500) antibodies (Abcam, London, UK) in 3% casein TTBS at 4°C after the samples were electrophoresed and transferred onto polyvinylidene fluoride membranes (Millipore), blocked and rinsed with 3% casein in TTBS. The membranes were rinsed and the SuperSignal® West Dura Extended Duration Substrate (Pierce) was used to detect each membrane incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Pierce) in 2% casein TTBS, and the density of each band was quantified thrice using the densitometry function of Bands can 5.0 software.
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