Following the drug treatments, cells were collected and resuspended in RIPA lysis buffer (Beyotime, China) and lysed by ultrasonic instrument. Then equal protein amount of the cell extracts was analyzed on a 12% SDS-polyacrylamide gels and transferred onto PVDF membranes. Membranes were blotted with primary antibodies targeting the following proteins: LSD1 (Sangon Biotech, Shanghai, China), pan-acetyl-H3 (Millipore, USA), Mono-Methyl-Histone H3 (Lys4), Di-Methyl-Histone H3 (Lys4), Tri-Methyl-Histone H3 (Lys4), Mono-Methyl-Histone H3 (Lys9), Di-Methyl-Histone H3 (Lys9), Tri-Methyl-Histone H3 (Lys9), PARP [poly(ADP-ribose) polymerase], Caspases-3, P21, P27, P57, Bcl-xL, Bcl-2, Bax and LC3B (all from Cell Signaling Technology, USA). β-actin (Abcam, USA) and Histone H3 (Abcam, USA) were used as internal controls. Then, membranes were incubated in HRP-conjugated secondary antibody and visualized using the HRP Substrate (Millipore, USA) by Image Quant LAS-4010 system (GE Healthcare, USA).
Caspases 3
Manufactured by Cell Signaling Technology
Sourced in United States
Caspases-3 is a lab equipment product that functions as a protease enzyme involved in the process of apoptosis, or programmed cell death. It plays a central role in the execution of this cellular process.
Automatically generated - may contain errors
Lab products found in correlation
Pan acetyl h3, by Merck Group (2 mentions)
Bcl xl, by Cell Signaling Technology (2 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (2 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Hrp substrate, by Merck Group (1 mentions)
Imagequant las 4010 system, by GE Healthcare (1 mentions)
Di methyl histone h3, by Cell Signaling Technology (1 mentions)
Tri methyl histone h3, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
β actin, by Abcam (1 mentions)
Histone h3, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Cytiva (1 mentions)
Anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Western blot detection kit, by Bio-Rad (1 mentions)
Anti rat igg, by Thermo Fisher Scientific (1 mentions)
Bcl2 associated x (bax), by Novus Biologicals (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh sc 32233, by Santa Cruz Biotechnology (1 mentions)
Caspase 9, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
3 protocols using caspases 3
1
Epigenetic and Apoptotic Pathway Analysis
2
Immunoblot Analysis of Apoptotic Proteins
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Immunoblot analysis was used to investigate cell apoptotic signal proteins. RIPA buffer (Thermo Fisher Scientific) containing a protease inhibitor cocktail (Roche, Basel, Switzerland) was used to extract total proteins, and the concentration of proteins were then measured using the Bradford assay. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the proteins were separated and then transferred to a polyvinylidene fluoride membrane (Amersham Bioscience, Uppsala, Sweden). The membranes were incubated with primary antibody, including anti-human poly (ADP-ribose) polymerase (PARP, #9542 s; Cell Signalling, Danvers, MA), caspases-3 (#9665 s; Cell Signalling), caspase-9 (#9508 s; Cell Signalling), bax (NB100–56095; Novus Biologicals, Centennial, CO), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-32233; Santa Cruz), at 4 °C overnight. After vigorous washing with tris-buffered saline, the horseradish peroxidase-conjugated secondary antibodies, such as anti-rabbit IgG or anti-rat IgG (Invitrogen, Waltham, MA), were applied to the membranes at room temperature for 1 hrs. The specific bands were developed using the ImageQuant LAS 4000 chemiluminescence imaging equipment (GE Healthcare, Chicago, IL) and a western blot detection kit from Bio-Rad (Hercules, CA).
3
Protein Extraction and Analysis
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Harvested cells were resuspended in a lysis buffer containing 50 mM Tris/HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS and 0.1 mM PMSF. Proteins were analyzed by SDS-PAGE and Western blotting. Membranes were incubated with primary antibodies against pan-acetyl-H3 (Millipore, USA), PARP [poly(ADP-ribose) polymerase], γ-H2AX (Ser139), caspases-3, Bcl-xL, Bcl-2, Bax, H3, β-actin (all from Cell Signaling Technology, USA). Blots were visualized using the ECL (Millipore, USA) with Image Quant LAS-4010 (GE Healthcare, USA).
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