Immunoblotting analysis was performed as described previously [21 (link)] using the following primary antibodies: caspase‐3 (1 : 1000; Cell Signaling Technology Inc., Danvers, MA, USA), BCL2‐associated X (BAX; 1 : 1000; Abcam, Cambridge, UK), caspase‐1 (1 : 1000; Abcam), caspase‐11 (1 : 1000; Abcam), GSDMD (1 : 1000; Abcam), NLRP3 (1 : 1000; Cell Signaling Technology Inc.), LC3‐B (1 : 1000; Cell Signaling Technology Inc.), mixed lineage kinase domain‐like protein (MLKL; 1 : 1000; Cell Signaling Technology Inc.), glutathione peroxidase 4 (GPX4; 1 : 1000; Cell Signaling Technology Inc.), proliferating cell nuclear antigen (PCNA; 1 : 1000; Cell Signaling Technology Inc.), and cyclin D1 (1 : 1000; Abcam). GAPDH was used as an internal control.
Bcl2 associated x bax
Manufactured by Abcam
Sourced in United States
BAX is a pro-apoptotic protein that belongs to the Bcl-2 family of proteins. It plays a key role in the intrinsic apoptosis pathway, promoting the release of cytochrome c from the mitochondria and the subsequent activation of caspases, leading to cell death.
Automatically generated - may contain errors
Lab products found in correlation
Cyclin d1, by Abcam (2 mentions)
B cell lymphoma 2 bcl 2, by Abcam (2 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Glutathione peroxidase 4 gpx4, by Cell Signaling Technology (1 mentions)
Gsdmd, by Abcam (1 mentions)
Caspase 11, by Abcam (1 mentions)
Mixed lineage kinase domain like protein mlkl, by Cell Signaling Technology (1 mentions)
Nlrp3, by Cell Signaling Technology (1 mentions)
Caspase 1, by Abcam (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Abcam (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Caspase 3 assay kit, by Abcam (1 mentions)
Apoptosis kits, by BD (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
N acetyl l cysteine, by Beyotime (1 mentions)
Phosphorylated pi3 k p pi3 k, by Abcam (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Cell Signaling Technology (1 mentions)
5 protocols using bcl2 associated x bax
1
Immunoblotting Analysis of Cell Death Markers
2
Western Blot Analysis of Apoptosis Markers
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Total protein extracted from lung tissue and HBECs was separated by sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. A 5% non-fat milk in Tris-buffered Saline with Tween (TBST) was then applied to block PVDF membranes for one hour at room temperature. Primary antibodies against B-cell lymphoma-2 (Bcl-2) (Cell Signaling Technology, USA, 1:1000), Bcl-2 Associated X (BAX) (Abcam, USA, 1:1000), Cleaved-caspase 3 (Cell Signaling Technology, USA, 1:1000), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Abcam, USA, 1:1000) were incubated with PVDF membranes overnight at 4°C. Then HRP-labeled IgG secondary antibodies (Proteintech, Wuhan, China, 1:5000) were incubated with PVDF members for one hour at room temperature. For protein band densities, ImageJ software was applied to analyze quantitatively.
3
Celastrol-Induced Apoptosis Regulation
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Celastrol was supplied by Baoji Herbest Bio-Tech Co., Ltd. Media were provided by Invitrogen (America). Antisubstances of ATF4, phosphorylated eukaryotic initiation factor 2 alpha (P-EIF2α), EIF2α, STAT3, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were supplied by Cell Signaling Technology (USA). Antisubstances, P-STAT3 and BCL2 Associated X (BAX), were provided by Abcam Co. (USA). Antibodies of B-cell lymphoma-2 (BCL-2) and secondary antibodies were purchased from Santa Cruz Biotechnology Inc. (USA). The Overall Protein Abstraction Kit was supplied by Boster Biological Technology (PRC). Dimethylsulfoxide (DMSO) and methylthiazolyldiphenyltetrazolium bromide (MTT) were provided by Sigma-Aldrich Co. (USA). Apoptosis kits were purchased from BD Pharmingen (USA). The Caspase 3 Assay Kit was purchased from Abcam Co. (Cambridge, MA, USA). ROS probes 2′,7′-Dichlorodihydrofluorescin diacetate (DCFH-DA) and N-Acetyl-l -cysteine (NAC) were obtained from Beyotime Biotech (Nantong, China).
4
Western Blot Analysis of Liver and Cell Proteins
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Briefly, the proteins extracted from the livers and cells were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to nitrocellulose filter membranes. After blocking in the protein-free rapid blocking buffer (Epizyme Biotech, Shanghai, China), the membranes were washed with tris-buffered saline + Tween 20 (TBST) for three times and incubated overnight with primary antibodies for FGF5 (1:500, Affinity Biosciences, Jiangsu, China), phosphorylated-PI3K (p-PI3K, 1:1000, Abcam), PI3K (p85, 1:1000, Abcam), phosphorylated-AKT (p-AKT; Ser473, 1:1000, Cell Signaling Technology, Danvers, MA, USA), AKT (1:1000, Cell Signaling Technology), B cell lymphoma-2 (BCL-2) (1:1000, Abcam), BCL2-associated X (BAX) (1:1000, Abcam), caspase-9 (1:1000, Abcam), cleaved-caspase-9 (1:1000, Abcam), caspase-3 (1:1000, Abcam), cleaved-caspase-3 (1:1000, Abcam), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1000, Cell Signaling Technology). The next day, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Finally, the visualization of membranes was realized by enhanced chemiluminescence reagent and in a ChemiDoc XRS+ system (BIO-RAD, Hercules, CA, USA). The gray value of protein was quantified by Image J software (National Institutes of Health, Bethesda, MD, USA).
5
Apoptosis Analysis of HDAC and XPO1 Inhibitors
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Tucidinostat (dissolved in DMSO as 50mg/ml) and selinexor (dissolved in DMSO as 50mg/ml) were obtained commercially from Selleckchem (Houston, TX, USA). Annexin V-FITC apoptosis detection kit was obtained from eBioscience (San Diego, CA, USA). The antibodies of HDAC1, HDAC 2, HDAC3, XPO1, acetyl-p53, p53, p21, Cyclin D1, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X (Bax) and GAPDH were purchased from abcam (Cambridge, UK).
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