Flash frozen hearts were homogenized at 4 °C in radio-immunoprecipitation assay (RIPA) buffer (in mmol·L−1: Tris 10, NaCl 140, EDTA 5, phenylmethanesulfonylfluoride PMSF 1 with Triton X-100 1%, deoxycholate 1%, sodium dodecyl sulfate (SDS) 0.1%, and in μg/mL: aprotinin 10, leupeptin 10, pepstatin 10, at pH 7.4) using a glass dounce tissue grinder. A mitochondria isolation kit for tissue (Abcam, Paris, France) was used to prepare enriched mitochondrial fractions, which were stored at −80 °C until use. Proteins (25 μg) were resolved by SDS-polyacrylamide gel electrophoresis (PAGE), transferred onto a nitrocellulose membrane and incubated with the following antibodies: rabbit polyclonal anti VDAC-1 antibody (1/1000; Abcam, Paris, France), rabbit polyclonal anti-phospho-Ser-58 CcOX IV-1 antibodies (1/500; Phospho Solutions, Aurora, CO, USA). Blots were developed with enhanced chemiluminescence (ECL) Plus reagent with goat anti-rabbit IgG antibody conjugated to horseradish peroxidase for chemiluminescent detection (Cell Signaling Technology, Beverly, MA, USA).
Anti vdac1 antibody
Manufactured by Abcam
Sourced in United States, United Kingdom
The Anti-VDAC1 antibody is a laboratory reagent used for the detection and study of the VDAC1 protein. VDAC1, also known as the voltage-dependent anion channel 1, is a protein involved in the regulation of mitochondrial function. This antibody can be used in various experimental techniques, such as Western blotting, immunohistochemistry, and immunoprecipitation, to identify and quantify the expression of VDAC1 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Adenosine triphosphate (atp), by Merck Group (3 mentions)
Anti mouse il 1β antibody, by R&D Systems (3 mentions)
Mitosox, by Thermo Fisher Scientific (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Nigericin, by Merck Group (2 mentions)
Mitochondria isolation kit for tissue, by Abcam (1 mentions)
Goat anti rabbit igg antibody conjugated to horseradish peroxidase, by Cell Signaling Technology (1 mentions)
N acetyl l cysteine nac, by Beyotime (1 mentions)
Gadph, by Cell Signaling Technology (1 mentions)
Mdivi 1, by MedChemExpress (1 mentions)
Lc3i 2, by Cell Signaling Technology (1 mentions)
Ro 3306, by Selleck Chemicals (1 mentions)
P drp1, by Cell Signaling Technology (1 mentions)
Mitotracker red, by Thermo Fisher Scientific (1 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
Poly da dt, by Merck Group (1 mentions)
Anti human caspase 1 antibody, by Santa Cruz Biotechnology (1 mentions)
Phospho stat1, by Cell Signaling Technology (1 mentions)
Mitotracker green, by Thermo Fisher Scientific (1 mentions)
Mouse anti caspase 1 p20, by Adipogen (1 mentions)
7 protocols using anti vdac1 antibody
1
Mitochondrial Protein Isolation and Western Blot
2
Mitochondrial Regulation and Cell Cycle Inhibition
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N-acetyl-L-cysteine (NAC) (Beyotime, Hangzhou, China); Mdivi-1 (MedChemExpress, Monmouth Junction, NJ, USA), a selective cell-permeable inhibitor of mitochondrial division; and RO-3306 (Selleck Chemicals LLC, Houston, TX, USA), a selective CDK1 inhibitor were used in their respective experiments. Antibodies against β-actin, caspase 3A/B, PINK1, LC3Ⅰ/Ⅱ, GADPH, CDK1 (cdc2), and Drp1 and its phosphorylated form (p-Drp1) were purchased from Cell Signaling Technology (CST, Boston, USA). Anti-VDAC1 antibody was from Abcam and the Anti-Parkin antibody was from Proteintech (Chicago, USA). Mouse anti-Cap monoclonal antibody was produced in our laboratory. Goat anti-rabbit or goat anti-mouse antibodies conjugated to horseradish peroxidase (HRP) were purchased from KPL (Gaithersburg, MD, USA).
3
Immunomodulatory Cytokine and Inflammasome Assays
4
Inflammasome Activation in Murine Macrophages
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LPS, ATP, nigericin, cycloheximide (CHX), CCCP, mdivi-1, propidium iodide (PI), U0126, and Drp1-targeting or non-targeting shRNA lentiviral plasmids were purchased from Sigma. Alum was purchased from InvivoGen. Ac-YVAD-chloromethylketone (Ac-YVAD-cmk) and z-VAD-fluoromethylketone (zVAD-fmk) were obtained from Bachem. Mouse IL-1β enzyme-linked immunoassay (ELISA) kits were obtained from R&D. MitoSOX, MitoTracker Green, MitoTracker Deep Red, and mouse recombinant TNF-α were purchased from Invitrogen. Annexin V-FITC apoptosis detection kit was obtained from BD Bioscience. Anti-human caspase-1 (p10), anti-ASC, anti-phospho-ERK and anti-β-actin antibodies were purchased from Santa Cruz. Anti-mouse IL-1β antibody was obtained from R&D. Anti-mouse caspase-1 (p20) and anti-NLRP3 antibodies were from Adipogen. Anti-Drp1 and anti-JNK1/2 antibody were purchased from BD Biosciences. Anti-phospho-JNK1/2 antibody was obtained from Invitrogen. Anti-VDAC1 antibody was obtained from Abcam. All other antibodies detecting human IL-1β, caspase-3, ERK, and IκB were obtained from Cell Signaling.
5
Identifying VDAC1 Oligomeric Forms
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To decipher the oligomeric forms of VDAC1, chemical crosslinking was performed in liver tissues and cells using the membrane permeable cross-linker EGS [ethylene glycol bis (succinimidyl succinate), Thermo Fisher Scientific Inc., Waltham, MA, USA] to stabilize oligomers. Briefly, cells were harvested by scraping and rinsed twice with PBS. Cells or protein lysates of liver tissue were incubated with 0.5 mM EGS in PBS, pH 7.4, at 30 °C for 20 minutes. To remove possible excess cross-linkers, 1.5 M Tris HCl (pH 7.8) was supplied to a final concentration of 20 mM, and the mixture was incubated for 5 minutes at room temperature prior to centrifugation at 10,000 × g for 5 minutes. The pellets were lysed in NP-40 lysis buffer by sonication on ice. The protein content was measured using the Pierce BCA Protein Assay (#P0011, Beyotime, Shanghai, China). The samples (50 µg) were then subjected to SDS‒PAGE and immunoblotting using an anti-VDAC1 antibody (#154856, Abcam, Cambridge, UK)14 (link),31 (link).
6
Mitochondrial Dysfunction and Proteostasis
7
Inflammasome Activation Pathway Analysis
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FK866, lipopolysaccharide (LPS), nigericin, ATP, poly (dA:dT), poly (I:C) and nicotinamide mononucleotide (NMN) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Flagellin purified from P. aeruginosa was obtained from In vivoGen (San Diego, CA, USA). FK866 used in the in vivo experiments was purchased from Cayman (Ann arbor, MI, USA). Ciliobrevin D was obtained from Calbiochem (San Diego, CA, USA). Anti-mouse caspase-1 and anti-mouse NLRP3 antibodies were purchased from Adipogen (San Diego, CA, USA). Anti-apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) antibody was purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-mouse IL-1β antibody was obtained from R&D Systems (Minneapolis, MN, USA). Anti-mouse gasdermin D (GSDMD) and anti-VDAC1 antibodies were purchased from Abcam (Cambridge, MA, USA). Anti-mouse β-actin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
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