A375 and A875 cells were harvested with RIPA buffer (Thermo Fisher Scientific) in compliance with the manufacturer’s instructions. Then, the protein concentration was examined using BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amount protein samples were loaded on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. Subsequently, membranes were blocked with 1% bovine serum albumin for 1 h at room temperature and probed with primary antibodies at 4°C overnight, and GAPDH (1:2000 dilution; Abcam, Cambridge, MA, USA) was used for normalization. After extensive washing, the membranes were incubated with a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:2000 dilution; Abcam) for 1 h. Finally, protein bands were visualized and analyzed with enhanced chemiluminescent method and Image J software (National Institutes of Health), respectively. The primary antibodies were listed as follow: hexokinase 2 (HK2; 1:1000 dilution; Abcam), E-cadherin (1:1000 dilution; Abcam), vimentin (1:1000 dilution; Abcam), N-cadherin (1:1000 dilution; Abcam), ITGA9 (1:1000 dilution; Abcam). ProliferatingCellNuclearAntigen (PCNA; 1:1000 dilution; Abcam), cyclin D1 (1:1000 dilution; Abcam), B-cell lymphoma-2 (Bcl-2; 1:1000 dilution; Abcam).
B cell lymphoma 2 bcl 2
Manufactured by Abcam
Sourced in United States, United Kingdom
Bcl-2 is a lab equipment product that plays a key role in the regulation of apoptosis, or programmed cell death. It functions as an anti-apoptotic protein, helping to prevent cell death. The Bcl-2 protein is involved in the mitochondrial pathway of apoptosis and is considered a central regulator of this process.
Automatically generated - may contain errors
Lab products found in correlation
Bcl 2 associated x protein bax, by Abcam (6 mentions)
Gapdh, by Abcam (5 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Proliferating cell nuclear antigen (pcna), by Abcam (2 mentions)
Vimentin, by Abcam (2 mentions)
E cadherin, by Abcam (2 mentions)
Cyclin d1, by Abcam (2 mentions)
Caspase 3, by Abcam (2 mentions)
P akt, by Abcam (2 mentions)
Cleaved caspase 3, by Abcam (2 mentions)
Caspase 9, by Abcam (2 mentions)
Bcl2 associated x bax, by Abcam (2 mentions)
Horseradish peroxidase conjugated goat anti rabbit secondary antibody, by Abcam (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Hexokinase 2 hk2, by Abcam (1 mentions)
N cadherin, by Abcam (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
P nf κb, by Abcam (1 mentions)
Phosphorylated akt, by Abcam (1 mentions)
Nf κb, by Abcam (1 mentions)
12 protocols using b cell lymphoma 2 bcl 2
1
Western Blot Analysis of Protein Expression
2
Spinal Cord Protein Expression Analysis
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The spinal cord tissues were taken out from liquid nitrogen, lysed in RIPA lysis buffer, containing protease inhibitor, for 30 min and centrifuged to obtain the supernatant which was used to determine the protein concentration. After SDS-PAGE, the proteins were transferred onto a membrane, sealed and incubated overnight at 4°C with antibodies against NF-κB (Abcam, USA), p-NF-κB (Abcam, USA), B-cell lymphoma-2 (Bcl-2) (Abcam, USA), Bcl-2-associated X protein (Bax) (Abcam, USA), phosphatidylinositol 3-kinase (PI3K) (Abcam, USA), protein kinase B (Akt) (Abcam, USA), phosphorylated (p)-Akt (Abcam, USA), heme oxygenase-1 (HO-1) (Abcam, USA), Nrf2 (Abcam, USA), trithorax-1 (TRX-1) (Abcam, USA), Raf-1 (Abcam, USA), MEK (Abcam, USA), ERK (Abcam, USA), p-MEK (Abcam, USA), p-ERK (Abcam, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Abcam, USA) (diluted at 1:1000). Next, the PVDF membrane (Sigma, USA) was cleansed in TBST and incubated with horseradish peroxidase-labeled secondary antibodies at room temperature for 2 h. Finally, the color of the proteins was developed using an ECL kit and gel imaging system, and the absorbance analyzed by Image Tools.
3
Protein Extraction and Western Blot Analysis
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Total proteins were extracted using RIPA buffer (Beyotime) and quantified using BCA Kit (Beyotime). The equivalent amount of protein was isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Then, the membranes were closed with 5% non-fat milk and incubated with the primary antibodies against caspase 3 (1:1000, Beyotime), B-cell lymphoma-2 (Bcl-2; 1:2000, Abcam, Cambridge, MA, USA), Bcl-2-associated x protein (Bax; 1:5000, Abcam), E-cadherin (1:1000, Abcam), Vimentin (1:2000, Abcam), FOXP2 (1:1000, Abcam), proliferating cell nuclear antigen (PCNA; 1:5000, Abcam) or GAPDH (1:5000, Abcam) at 4°C overnight. After incubated with the secondary antibody (1:2000, Abcam) for 1 h, the membranes were treated with enhanced chemiluminescence reagent (Beyotime) to visualize the protein signals.
4
Western Blot Protein Analysis
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Cells at 80% confluence were washed three times with PBS. RIPA buffer was added to extract the total protein, and equal amounts of protein were loaded onto 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels for protein separation. Then, proteins were transferred onto nitrocellulose membranes. The membranes were blocked with 5% nonfat dried milk for 3 h and incubated with primary antibodies (p53 at 1:1000, p-AKT [CST, USA] at 1:800, total AKT [CST, USA] at 1:800, B-cell lymphoma-2 [Bcl-2] [Abcam, UK] at 1:1000, Bcl-2 associated X protein [Bax] [Abcam, UK] at 1:1000, caspase-3 [Abcam, UK] at 1:1000, and iNOS [CST, USA] at 1:1,000) overnight at 4°C. After being washed in Tris-buffered saline (pH 7.2) containing 0.05% Tween 20, the membranes were incubated with the secondary antibody for 2 h at room temperature. Finally, the membranes were washed, and ECL chemiluminescence reagents (Amersham Pharmacia Biotech, Japan) were used to detect the proteins. The quantification of band intensity was performed using Quantity One software (Bio-Rad, Hercules, CA, USA).
5
Signaling Pathways in Cardioprotection
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DOX was purchased from Haizheng Pfizer Pharmaceutical Co., Ltd. Levosimendan was obtained from Orion Corporation, Espoo, Finland. The following primary antibodies were purchased from Cell Signaling Technology (Boston, MA, USA): Bcl-2-associated X protein (BAX; 1 : 1000), c-caspase-3 (1 : 1000), PTEN (1 : 1000), P-Akt (1 : 1000), T-Akt (1 : 1000), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1 : 1000), and B-cell lymphoma-2 (Bcl-2) (1 : 1000) was purchased from Abcam. Akt inhibitor (Akt i) was purchased from Sigma-Aldrich (St. Louis, MO, USA). A goat anti-rabbit secondary antibody was purchased from LI-COR Biosciences (Lincoln, USA). The BCA protein assay kit was obtained from Dōjindo Laboratories (Kumamoto, Japan).
6
Mep-S Neuroprotective Mechanism Protocol
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Mep-S was synthesized in our laboratory as previously described [19 (link)]. Scopolamine, H2O2, and N-acetylcysteine (NAC) were the products of Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM)/F-12 (1 : 1), phosphate-buffered saline (PBS), and a mixture of penicillin and streptomycin were the products of Gibco (Grand Island, NY, USA). The rabbit-derived antibodies against NAD(P)H quinine oxidoreductase-1 (NQO-1; cat #ab80588), heme oxygenase-1 (HO-1), Kelch-like ECH-associated protein-1 (Keap1), Nrf2, phospho-Nrf2 (S40), and B-cell lymphoma 2 (Bcl-2) were the products of Abcam (Cambridge, MA, USA). The rabbit-derived antibodies against caspase-3 (D3R6Y), phospho-Akt (Ser473), Akt, and Bax were the products of Cell Signaling Technology (CST; Danvers, MA, USA). Mep-S was dissolved in dimethyl sulfoxide (DMSO; Sigma), and NAC was dissolved in normal saline to obtain stock solutions (20 mg/ml), which were diluted with saline (for in vivo experiments) or culture medium (for in vitro tests). The Mep-S working solutions contain less than 2.5% of DMSO.
7
Western Blot Analysis of Cellular Signaling
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A lysis buffer (RIPA, Beyotime, China) was added to each group of cells or tissues to fully lyse and extract total protein from the cells. Lysates were electrophoresed on 6-12% gels using sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE). Protein isolates were transferred to PVDF membranes (Millipore, USA) and blocked in 5% skim milk powder solution for 1 h at room temperature. The cells were incubated with primary antibody overnight at 4°C. Primary antibodies against GPX4 (Proteintech, USA), B-cell lymphoma-2 (Bcl-2) (Abcam, USA), Cleaved Caspase-3 (CST, USA), p-ERK (CST, USA), ERK (CST, USA), p-p38 (CST, USA), p38 (CST, USA), p-JNK (CST, USA), JNK (CST, USA), and β-actin (Santa Cruz, USA) were used. The appropriate secondary antibody (1 : 5000; Cell Signaling Technology, USA) was incubated for 1 hour at 37°C on a shaker, and the bands were observed using ECL chemiluminescence (Beyotime Biotechnology, China). ImageLab software was used for data analysis.
8
Protein Expression Analysis in Apoptosis
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The frozen lung tissue was homogenized (1:9, w/v) with a tissue lysis/extraction reagent (Sigma-Aldrich, St. Louis, MO, USA) containing a protease/phosphatase inhibitor cocktail (Sigma-Aldrich) and was centrifuged at 12,000 × g for 10 min at 4°C to isolate the cellular proteins in the supernatant. To investigate the protein expression levels related to apoptotic changes, we performed western blotting according to a previous study [21 (link)]. The following primary antibodies and dilutions were used: total phosphatidylinositol 3-kinase (PI3K; 1:1000 dilution; Abcam), phospho (p)-PI3K (1:1000 dilution; Abcam), total protein kinase B (AKT; 1:1000 dilution; Abcam), p-AKT (1:1000 dilution; Abcam), B-cell lymphoma 2 (Bcl-2; 1:1000 dilution; Abcam), Bcl-2-associated X protein (Bax; 1:1000 dilution; Abcam), β-actin (1:2000 dilution; Abcam), vascular endothelial growth factor (VEGF; 1:1000 dilution; Novus Biologicals, Littleton, CO, USA), and cleaved Caspase 3 (1:1000 dilution; Cell Signaling Technology). Relative protein expression levels were determined using Chemi-Doc (Bio-Rad Laboratories, Hercules, CA, USA).
9
Western Blot Analysis of Tissue Proteins
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Tissue proteins were extracted by RIPA lysis buffer (Cell Signaling Technology, USA) containing a protease inhibitor cocktail (KeyGEN BioTECH, China). The protein concentration was determined by the BCA protein assay (Beyotime, China). After that, the proteins were isolated by SDS–PAGE and then passed on to a PVDF membrane (Millipore, USA). The membrane was blocked with 5% bovine serum albumin at room temperature for 1 h and then incubated with primary antibodies against collagen I (Abcam, USA), matrix metalloproteinase 9 (MMP9) (Abcam, USA), IGFBP3 (Abcam, USA), B-cell lymphoma-2 (Bcl-2) (Abcam, USA), Bcl-2-associated X protein (Bax) (Abcam, USA), and GAPDH (Abcam, USA). The membrane was washed and soaked with appropriate secondary antibodies at room temperature for 1 h. The protein bands were visualized using enhanced chemiluminescence (ECL) reagents (Millipore, USA) and imaged with an imaging system (Tanon, China). The GAPDH signal was used as a loading control.
10
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).
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