Kidney tissues were subjected to western blot analysis using a procedure described previously.24 After blocking nonspecific binding with 5% nonfat milk in phosphate‐buffered saline solution or 1 h at room temperature, membranes were incubated with the primary antibodies overnight at 4°C. The primary antibodies include anti‐NDUFV1 antibody (PA5‐98007; Invitrogen), anti‐SDHA antibody (11,998; Cell Signalling Technology), anti‐HSP60 antibody (12,165; Cell Signalling Technology), anti‐PHB1 antibody (2426; Cell Signalling Technology), anti‐VDHC antibody (4661; Cell Signalling Technology), anti‐Cox IV antibody (4850; Cell Signalling Technology), anti‐Bax antibody (2772; Cell Signalling Technology), anti‐Bcl‐2 antibody (3498; Cell Signalling Technology), anti‐cleaved Caspase 3 antibody (9664; Cell Signalling Technology), and anti‐Actin antibody (3700; Cell Signalling Technology). After three times washing in Tris‐buffered saline with Tween (TBST), membranes were then incubated with an appropriate secondary antibody for 1 h at room temperature. Membranes were developed using a chemiluminescence reagent. The Image J software was used to analyse the densitometry of the blots.
Anti bcl 2 antibody
Manufactured by Cell Signaling Technology
Sourced in United States, China, United Kingdom
The Anti-Bcl-2 antibody is a laboratory reagent used in research applications. It is a protein that binds to the Bcl-2 protein, which plays a role in regulating cell survival and apoptosis. The antibody can be used to detect and quantify Bcl-2 expression in various cellular and biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti bax antibody, by Cell Signaling Technology (15 mentions)
Anti caspase 3 antibody, by Cell Signaling Technology (8 mentions)
Anti gapdh antibody, by Cell Signaling Technology (5 mentions)
Anti cleaved caspase 3 antibody, by Cell Signaling Technology (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Anti β actin antibody, by Santa Cruz Biotechnology (3 mentions)
Anti bcl xl antibody, by Cell Signaling Technology (3 mentions)
Anti parp antibody, by Cell Signaling Technology (3 mentions)
Anti erk1 2 antibody, by Cell Signaling Technology (3 mentions)
Anti mcl 1 antibody, by Cell Signaling Technology (2 mentions)
Anti p erk1 2 antibody, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti atf4 antibody, by Cell Signaling Technology (2 mentions)
Anti cox 4 antibody, by Cell Signaling Technology (1 mentions)
Anti actin antibody, by Cell Signaling Technology (1 mentions)
Anti hsp60 antibody, by Cell Signaling Technology (1 mentions)
Anti sdha antibody, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Anti mfn2 antibody, by Abcam (1 mentions)
Anti p21 antibody, by Santa Cruz Biotechnology (1 mentions)
33 protocols using anti bcl 2 antibody
1
Western Blot Analysis of Kidney Proteins
2
Western Blot Analysis of Mitochondrial Proteins
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The proteins (30μg) described above were then separated by SDS-PAGE, and transferred to PVDF membranes (Invitrogen, USA). Nonspecific binding was blocked by using of 5% nonfat milk for 1 h at 37°C. The nitrocellulose membranes were then hybridized overnight at 4°C with the rabbit polyclonal anti-Mfn2 antibody (Abcam, USA), the rabbit polyclonal anti-Bcl-2 antibody (Cell Signaling Technology, USA), the rabbit polyclonal anti-Bax antibody (Cell Signaling Technology, USA), and the rabbit polyclonal anti-β-actin antibody (Santa cruz Biotechnology Inc, USA) in the Primary Antibody Dilution Buffer. After washing four times with TBS-T, each time for 10 minutes at 37°C the membranes were incubated with goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at 37°C (AntGene, USA). Finally, the immunoreactive bands were detected by use of the Enhanced Chimio-Luminescent system (Beyotime Institute of Biotechnology, China). Washing the bands four times in TBS-T. The band intensity was quantified by densitometry using the Quantity One 4.62 analysis software, and all results were normalized to β-actin signal intensity.
3
Protein Expression Analysis via Western Blot
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The Western blot analysis was performed using a previously described method.26 (link) The following antibodies were used: anti–Bcl-xL antibody, anti–Bcl-2 antibody, anti–Mcl-1 antibody (Cell Signaling Technology), anti-p21 antibody (Santa Cruz Biotechnology), and anti–β-actin antibody (Sigma-Aldrich, St. Louis, MO).
4
Comprehensive Protein Analysis Protocol
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Total-cell lysates were prepared using RIPA buffer (150 mM NaCl, 1%NP-40, 50 mM Tris-HCl (pH 7.4), 1 mM phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 mM Deoxycholic acid and 1 mM EDTA) containing a cocktail of protease inhibitors and phosphatase inhibitors (Calbiochem, Darmstadt, Germany). Equal amount of proteins (40∼60 μg) was separated by 12% SDS-PAGE and transferred to PVDF membrane (Millipore, Bedford, MA, USA) using the Bio-Rad semidry transfer system. The following antibodies were used for Western blot: Anti-RhoA antibody (Santa Cruz Biotechnologies, USA), Anti-GAPDH antibody, Anti-bcl2 antibody, Anti-bcl-xl antibody, Anti-bax antibody (Cell Signaling Technology, USA), Anti-ABCB1 antibody, Anti-ABCC1 antibody, Anti-GSPT1 antibody (Abcam, UK). Blotted proteins were detected and quantified using the ODYSSEY Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA).
5
Investigating Bcl-2 Ubiquitination in HEK Cells
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HEK cells were transfected with the indicated amount of PAK1-DN or PAK1-CA plasmids, incubated for 36 h, treated with 10 μM lactacystin (Tocris) for 12 h and subjected to immunoprecipitation with anti-Bcl-2 antibody (Cell Signaling). Immunoprecipitates were subjected to Western blot analysis using anti-monoclonal mono- and polyubiquitinylated conjugates antibody (FK2, BML-PW8810, Enzo Life Sciences, Farmingdale, NY, USA).
6
Immunoprecipitation of Bcl-2 and Beclin-1
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Briefly, homogenates (200 µg) were incubated with anti-Bcl-2 antibody (3 µg, Cell Signaling Technology, Beverly, MA, USA) at 4 °C overnight. Protein G-Sepharose beads (30 µl/tube, Sigma, St. Louis, MO, USA) were prewashed three times in immunoprecipitation (IP) buffer (10 mM Tris-Cl, pH 7.5, 150 mM sodium chloride, 2 mM EDTA, 0.5% Triton-100) for 15 min and incubated with a protein/antibody mixture under constant rotation at 4 °C for 2 h. The precipitant was collected by centrifugation at 10000 × g for 1 min and washed three times with IP buffer to remove nonspecifically bound proteins. Then, the immune-complexed beads, which were resuspended in SDS-PAGE loading buffer (60 µl/tube) and heated at 95 °C for 5 min, were removed by centrifugation at 10000 × g, and the supernatants were used for immunoblot detection of beclin-1 and Bcl-2. The left homogenates without IP were treated as the input controls.
7
Apoptosis Pathway Regulation Assay
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Dulbecco’s modified Eagle’s medium (DMEM), 1640 medium and diamidino-2-phenylindole (DAPI) were obtained from (Sigma Millipore, St. Louis, MO, USA). Fetal bovine serum (FBS) was acquired from GIBCO (Sigma Millipore, St. Louis, MO, USA). The BCA Protein Assay Kit was from Junyan Biotechnology (S0111A-96, Taiyuan, China). DSF (T0054) and Diethyldithiocarbamate-copper complex (CuET) was obtained from TOPSCIENCE (Shanghai, China). The Anti-Bax antibody was purchased from (2774, Cell signaling Technology, Danvers, MA, USA). The Anti-Bcl-2 antibody was purchased from (3498, Cell signaling Technology, Danvers, MA, USA). The Anti-cleaved caspase-3 antibody was purchased from (9661, Cell signaling Technology, Danvers, MA, USA). The anti-GAPDH antibody was obtained from (5174, Cell signaling Technology, Danvers, MA, USA).
8
Immunoblotting Analysis of Cell Signaling Proteins
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Total cell lysate was extracted in the RIPA buffer containing protease inhibitor cocktail as described previously [35 (link)]. 25 μg of lysate from each cell line was separated using SDS-PAGE gel and transferred to Immobilon P membrane (Millipore). Blots were probed with the following antibodies: anti-Bcl-2 antibody (2870, Cell Signaling, USA), anti-Bcl-xL (2764, Cell Signaling, USA), anti-phospho-Bcl-xL (sc-101644, Santa Cruz, USA), anti-survivin (2808, Cell Signaling, USA) and anti-actin monoclonal antibody (Sigma, USA) in blocking buffer (Li-Cor, NB, USA). Then the membranes were incubated with IRDye secondary antibodies (Licor, NB, USA). The membranes were scanned and the images were captured with Odyssey SA (Licor, NB, USA). Images were analyzed and quantified using ImageStudio software.
9
Western Blot Analysis of Myocardial Proteins
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The amount of 40 μg protein prepared from rat myocardium or NRVCs was used in a standard Western blot analysis. The polyvinylidene fluoride (PVDF) membrane binding sample protein was incubated with a high affinity anti-Hsp90aa1 antibody (1:2000 dilution), anti-Hsp90b1 antibody (1:1000) (Abcam, Cambridge, MA), anti-Bax antibody (1:1000), anti-Bcl-2 antibody (1:1000) and anti-Caspase-3 antibody (1:1000) (Cell Signaling Technology, Danvers, MA), respectively. An anti-β-actin antibody (1:2000) (Santa Cruz Biotechnology, Santa Cruz, CA) was used to detect level of β-actin as an internal control. Proteins were visualized using the ECL Plus detection system (GE Healthcare, Waukesha, WI).
10
Protein expression analysis by Western blot
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Protein extraction and western blot analysis were conducted as described before.31 The primary antibodies included: anti‐TFAM antibody (ab131607, 1:1000), anti‐ATPB antibody (ab14730, 1:5000), anti‐GPX4 antibody (ab125066, 1:3000) and anti‐HMGB1 antibody (ab79823, 1:10000) from Abcam (Cambridge, UK), anti‐BCL2 antibody (3498, 1:1000), anti‐LC3B antibody (3868, 1:1000) and anti‐P62 antibody (5114, 1:1000) from Cell Signaling Technology (Danvers, MA, USA), HRP‐conjugated mouse anti‐ACTB (KC‐5A08, 1:5000), and HRP‐conjugated mouse anti‐GAPDH (KC‐5G5, 1:5000) from Aksomics (Shanghai, China).
The HRP‐conjugated secondary antibodies involved anti‐mouse IgG (KC‐MM‐035, 1:5000) and anti‐rabbit IgG (KC‐RB‐035, 1:5000) from Aksomics (Shanghai, China). The protein bands were visualized with the ChemiDoc MP system (Bio‐Rad, CA). ImageJ software was applied to quantify the band intensity, and the results are presented as the ratio of the target protein to GAPDH or ATCB.
The HRP‐conjugated secondary antibodies involved anti‐mouse IgG (KC‐MM‐035, 1:5000) and anti‐rabbit IgG (KC‐RB‐035, 1:5000) from Aksomics (Shanghai, China). The protein bands were visualized with the ChemiDoc MP system (Bio‐Rad, CA). ImageJ software was applied to quantify the band intensity, and the results are presented as the ratio of the target protein to GAPDH or ATCB.
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