Cells were cultured with different concentrations of N69B for 48 h, then collected and lysed by RIPA buffer containing PMSF (Beyotime Biotechnology, Shanghai, China). Lysates were cleared by centrifugation to remove insolubles; protein concentrations were determined by the Enhanced BCA Protein Assay Kit (Beyotime Biotechnology). Forty micrograms of proteins were separated by SDS-PAGE and transferred onto polyvinylidene fluoride membrane (Merck Millipore). The membrane was blocked by 5% milk for 2 h, and then incubated with primary antibody at 4°C overnight. The membrane was washed three times, then incubated with secondary antibody at room temperature for 1 h. The bands were detected by Gel Imaging System (Bio-Rad, USA), and measured by ImageJ. The primary antibodies used were rabbit anti-β-actin, EGFR, bFGF, cathepsin D (CTSD), Bid, cytochrome C (Cyt C), caspase 3, caspase 8 (Cell Signaling Technology, USA), Bax, caspase 9, cleaved caspase 9 (Affinity Biosciences, USA)
Cleaved caspase 9
Manufactured by Affinity Biosciences
Sourced in United States, China
Cleaved caspase 9 is a laboratory reagent used in cellular and biochemical assays. It is an enzyme fragment generated by the activation of caspase 9, a key mediator of apoptosis. Cleaved caspase 9 can be used to detect and quantify apoptotic activity in various experimental models.
Automatically generated - may contain errors
Lab products found in correlation
Caspase 9, by Affinity Biosciences (1 mentions)
Cathepsin d, by Cell Signaling Technology (1 mentions)
Caspase 8, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Affinity Biosciences (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Gel imaging system, by Bio-Rad (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Enhanced bca protein assay kit, by Beyotime (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Cleaved caspase 3, by Affinity Biosciences (1 mentions)
Histone h3, by GeneTex (1 mentions)
P iκb, by Affinity Biosciences (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Bca protein assay kit, by Solarbio (1 mentions)
Nf κb, by ABclonal (1 mentions)
Goat anti rabbit igg hrp, by Solarbio (1 mentions)
2 protocols using cleaved caspase 9
1
Investigating N69B-induced Apoptotic Signaling
2
Protein Expression Analysis in Brain Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Proteins from brain tissues were extracted and quantified by bicinchoninic acid (BCA) protein assay kit (Solarbio, Beijing, China). Equal amounts of samples were separated by SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% skim milk for 1 h and incubated with the primary antibodies overnight at 4 ° C, including ROCK2 (1:1000, ABclonal, Wuhan, China), cleaved-caspase-3 (1:1000, Affinity, Changzhou, China), cleaved-caspase-9 (1:1000, Affinity, Changzhou, China), p-IκB (1:1000, Affinity, Changzhou, China), IκB (1:1000, Affinity, Changzhou, China), NF-κB (1:1000, ABclonal, Wuhan, China). β-actin (1:10,000, Santa Cruz, California, USA) and Histone H3 (1:5000, Gene Tex, Texas, USA) were used as internal controls. The membranes were incubated with goat anti-rabbit IgG-HRP or goat anti-mouse IgG-HRP secondary antibody (1:3000, Solarbio, Beijing, China) at 37 ° C for 1 h. The proteins were determined by an ECL western blot detection kit (Solarbio, Beijing, China).
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