The tissues and cells were smashed and homogenized with RIPA (Beyotime). After centrifugation, the supernatant containing protein was collected. Protein concentrations were measured by using the BCA protein concentration Kit (Beyotime). After 50 μg protein was denatured at 100˚C for 10 minutes, SDS‐PAGE electrophoresis was performed for protein separation, and then, the protein was transferred to PVDF membrane. The PVDF membrane was blocked by 5% skim milk for 1 hour and then incubated with specific primary antibodies, including anti‐APAF‐1 (1:1000; Abcam), anti‐cleaved caspase‐9 (1:1000; Abcam), anti‐cleaved caspase‐3 (1:1000; Abcam) and anti‐GAPDH (1:1000; Zhongshan Jinqiao Biotechnology), at 4°C overnight. The PVDF membrane was incubated with horseradish peroxidase labelled as goat anti‐rabbit or goat anti‐mouse immunoglobulin G (1:4000; EarthOx) at room temperature for 30 minutes. Detection of protein band was performed by using an enhanced chemiluminescence (ECL) for Western blotting kit (Beyotime) according to the manufacturer's instructions. Relative densitometry was calculated by using Image J2x analysis software.
Anti apaf 1
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-Apaf-1 is a primary antibody that binds to Apaf-1, a protein involved in the regulation of apoptosis (programmed cell death). This antibody can be used in various laboratory techniques, such as Western blotting, immunoprecipitation, and immunohistochemistry, to detect and study the expression and distribution of Apaf-1 in different cell types and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Anti cleaved caspase 9, by Abcam (2 mentions)
Anti cleaved caspase 3, by Abcam (2 mentions)
Enhanced chemiluminescence reagent, by Cell Signaling Technology (2 mentions)
Bca protein concentration kit, by Beyotime (1 mentions)
Anti gapdh, by Zhongshan Jinqiao Biotechnology (1 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
Ab32372, by Abcam (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Sc 40, by Santa Cruz Biotechnology (1 mentions)
Anti c myc, by Santa Cruz Biotechnology (1 mentions)
Pierce bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions)
Anti pro caspase 3, by Abcam (1 mentions)
Horseradish peroxidase labeled goat anti rabbit igg antibody, by Abcam (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Ripa lysis buffer, by Biosharp (1 mentions)
P ikk, by Cell Signaling Technology (1 mentions)
Traf2, by Cell Signaling Technology (1 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
7 protocols using anti apaf 1
1
Western Blot Analysis of Apoptosis Markers
2
Western Blot Analysis of Apoptosis Markers
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Samples of tissues and cultured cells were lysed in RIPA sample buffer and centrifuged at 12 000 × g for 10 min at 4°C. The supernatant fraction was collected, and the protein concentration was determined by a BCA assay (Pierce, Rockford, IL, USA). Proteins were fractionized on SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The membranes were blocked for 1 h, followed by an overnight incubation at 4°C with antibodies. After washes, membranes were incubated at room temperature for 1 h with the appropriate secondary antibody conjugated to horseradish peroxidase and detected with an enhanced chemiluminescence reagent (Cell Signaling Technology Inc., Danvers, MA, USA). The intensity of each band was scanned and quantified using BandScan software (Glyko Inc., Novato, CA, USA). The following antibodies were used: anti-Apaf-1 (rabbit, 1 : 500, Abcam, ab32372, Cambridge, MA, USA) and anti-cleaved caspase-3 (rabbit, 1 : 1000, Cell Signaling Technology Inc., 9661).
3
Western Blot Analysis of Apaf-1 and c-Myc
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Samples of tissues and cultured cells were lysed in RIPA sample buffer and centrifuged at 12000×g for 10 min at 4°C. The supernatant fraction was collected, and the protein concentration was quantified with a BCA assay (Pierce, Rockford, IL, USA). Proteins were separated on SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The membranes were blocked for 1 h, followed by an overnight incubation at 4°C with primary antibodies. After washing, the membranes were incubated at room temperature for 1 h with the appropriate secondary antibody conjugated to horseradish peroxidase and then detected with an enhanced chemiluminescence reagent (Cell Signaling Technology Inc., USA). The intensity of each band was scanned and quantified using BandScan software (Glyko Inc., Novato, CA, USA). The following antibodies were used: anti-c-Myc (1:200, sc-40, Santa Cruz Biotechnology, Inc.) and anti-Apaf-1 (1:500, ab32372, Abcam, Cambridge, MA, USA).
4
Western Blot Quantification of Apoptosis Markers
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Tissue proteins were obtained using RIPA lysis buffer (Biosharp). Protein concentrations were quantified by the Pierce Bicinchoninic Acid Protein Assay kit (Thermo Fisher Scientific). The proteins were transferred to the polyvinylidene fluoride membrane after being separated with a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and sealed in 5% skim milk and then incubated with primary antibody at 4°C overnight. All the primary antibodies we used were as follows: anti-APAF1 (1:2000; Abcam, Cambridge, UK), anti-clv-caspase-3 (1:2000; Abcam), anti-pro-caspase-3 (1:2000; Abcam), anti-N-GSDME (1:2000; Abcam), and anti- GAPDH (1:1000, Abcam). The membranes were then incubated with horseradish peroxidase-labeled goat anti-rabbit IgG antibody (1:5000; Abcam) for 1 h. The bands were examined using Tanon 5200 Automatic chemiluminescence image analysis system (Shanghai, China). Enhanced chemiluminescence solution was applied for color development to observe images.
5
Western Blot Analysis of Apoptosis Markers
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RIPA buffer (Beyotime, Nantong, China) was used to lyse cells, after which Western blot was conducted according to standard protocols using the following primary antibodies specific: GAPDH (Cat# 3683, Cell Signaling Technology, MA, USA), p-IKK (Cat# 2078), PARP (Cat# 5625), and TRAF2 (Cat# 2141). HRP-conjugated secondary antibodies were also purchased from Cell Signaling Technology, while anti-APAF1 (Cat# ab2001) was purchased from Abcam (Hercules, CA, USA).
6
Western Blot Analysis of ER Stress Markers
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RiPA buffer (GenStar, China) was used to extract the proteins from the transfected cell lines. The proteins were loaded and separated on SDS-PAGE, and transferred onto PVDF membranes (Millipore, USA). After blocking with 5% non-fat milk, the membranes were incubated with anti-ATF6 (Bioss, China), anti-EMC6 (Proteintech, USA), anti-APAF1 (Abcam, UK), and anti-GAPDH (Fude Biological Technology Co., Ltd. China) antibodies overnight at 4°C. Following this step, the membranes were incubated with horseradish peroxidase-coupled secondary antibodies for 1 h, and finally, the expression of the proteins was revealed using the enhanced chemiluminescence solution (ECL, PerkinElmer, USA).
7
Apoptosis and Wnt Signaling Pathway Analysis
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Protein samples were collected from cell lysates and protein concentrations were determined using a BCA kit (Beyotime). Proteins were separated by 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis electrophoresis and transferred to a nitrocellulose membrane. The membrane was blocked with 5% Tris-buffered saline with Tween 20 for 2 hours at room temperature and then incubated with primary antibody overnight at 4°C. The primary antibodies were: anti-caspase-3, anti-cleaved caspase-3, anti-caspase-9, anti-cleaved caspase-9, anti-BAX, anti-Bcl-2, anti-CytC, anti-Apaf-1, anti-COX IV, anti-PAX2, anti-GSK-3β, anti-pGSK-3β(Ser9), anti-β-catenin, anti-p-β-catenin(Ser33+Ser37), anti-Wnt2, anti-Wnt4, anti-Wnt5a, anti-Wnt10b, anti-Wnt11, anti-Wnt13, anti-Wnt14 (Abcam, Cambridge, UK), anti-GAPDH, and anti-β-actin (Cell Signaling Technology, Danvers, MA, USA). Films were cleaned 3 times with TBST and incubated with the corresponding horseradish peroxidase-conjugated secondary antibody for 1 hour. Membranes were visualised using the enhanced chemiluminescence reagents (Millipore, Burlington, MA, USA), and then the blots were quantified using ChemiDoc XRS system (Bio-Rad, Hercules, CA, USA).
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