The antibodies used in this study were as follows: anti-P2X7 (Abcam, Cambridge, UK; cat. no. ab109054), anti-collagen II (Abcam; cat. no. ab34712), anti-MMP13 (Abcam; cat. no. ab39012), anti-AMPKα1 (Abcam; cat. no. ab32047), anti-mTOR (Abcam; cat. no. ab109268), anti-NLRP3 (Proteintech; cat. no. 19771-1-AP), anti-caspase-1 (Proteintech; cat. no. 22915-1-AP), anti-LC3B (Abcam; cat. no. ab192890), anti-Beclin-1 (Abcam; cat. no. ab62557), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Proteintech; cat. no. 10494-1-AP), and horseradish peroxidase (HRP)-labeled IgG (Beyotime; cat. no. A0208). The reagents used in the experiment were as follows: MIA (Sigma, St. Louis, MO, USA; cat. no. I2512), the P2X7 receptor agonist BzATP (Sigma; cat. no. B6396), the mTOR activator MHY1485 (Sigma; cat. no. SML0810), the mTOR inhibitor rapamycin (Sigma; cat. no. V900930), the NLRP3 inhibitor CY-09 (Sigma; cat. no. SML2465), the AMPK activator A-769662 (Sigma; cat. no. SML2578), and the AMPK inhibitor compound C (Sigma; cat. no. P5499). The concentrations, dosages, and preparation of the reagents were described previously [43 (link), 45 (link)].
Anti p2x7
Manufactured by Abcam
Sourced in United Kingdom
Anti-P2X7 is a protein that acts as a receptor for the extracellular signaling molecule ATP. It is involved in the regulation of cellular processes such as inflammation and cell death.
Automatically generated - may contain errors
Lab products found in correlation
Bzatp, by Merck Group (2 mentions)
Anti nlrp3, by Proteintech (2 mentions)
Anti collagen 2, by Abcam (2 mentions)
Anti mmp13, by Abcam (2 mentions)
Anti caspase 1, by Proteintech (2 mentions)
Compound c, by Merck Group (1 mentions)
Rapamycin, by Merck Group (1 mentions)
Mhy1485, by Merck Group (1 mentions)
Anti beclin1, by Abcam (1 mentions)
Anti lc3b, by Abcam (1 mentions)
Anti ampkα1, by Abcam (1 mentions)
Anti glyceraldehyde 3 phosphate dehydrogenase gapdh, by Proteintech (1 mentions)
A 769662, by Merck Group (1 mentions)
Anti mtor, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Chemiluminescent hrp substrate, by Merck Group (1 mentions)
Nf κb p65, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Proteintech (1 mentions)
4 protocols using anti p2x7
1
Investigating Molecular Pathways in Osteoarthritis
2
Zinc Oxide Nanoparticle-Induced Signaling
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After treatmenting with ZnO NPs, cells were washed twice with PBS, and then RIPA lysis buffer (Beyotime, Haimen, China) with protease inhibitor and phosphatase inhibitors was added to extract the protein. The bicinchoninic acid (BCA) protein assay (Pierce BCA Protein Assay Kit; Thermo Fisher Scientifc, USA) was used to determine protein concentration. An equal amount of protein was separated using 8% SDS-PAGE. The proteins were transferred to polyvinylidene difluoride membranes (Merck Millipore, Darmstadt, Germany) and blocked with TBST containing 5% skim milk for 1 h at room temperature and then incubated overnight at 4 °C with specific antibodies including: anti-P2X7 (1:1000, abcam), NF-κB p65, phospho-NF-κB p65, p44/42 MAPK, phospho- p44/42 MAPK, p38 MAPK, phospho-p38 MAPK (1:1000, CST) and GAPDH (1:1000, Proteintech, USA). The blots were then washed by TBST, exposed to HRP-conjugated secondary antibodies (1:1000, CST, USA) for 1 h, and the antigen-antibody complex was detected with chemiluminescent HRP substrate (Millipore) by a chemiluminescence measuring instrument (Tanon 5200, Shanghai, China).
3
Antibody-based Western Blot Analysis
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The following antibodies were employed for western blotting: anti-P2X7 (Abcam, Cambridge, UK), anti-collagen II (Abcam), anti-MMP13 (Abcam), anti-IL-1β (Abcam), anti-NF-κB p65 (Cell Signaling Technology (CST), Danvers, MA, USA), anti-p-NF-κB p65 (CST), anti-NLRP3 (Proteintech Group, Chicago, IL, USA), anti-caspase-1 (Proteintech Group), anti-GAPDH (Proteintech Group), and HRP-labeled IgG (Beyotime Biotech, Shanghai, China). Primary chondrocyte cultures were treated with MIA (Sigma-Aldrich, St. Louis, MO, USA), P2X7R antagonist A740003 (Sigma-Aldrich), P2X7R agonist BzATP (Sigma-Aldrich), NF-κB inhibitor Bay 11-7082 (Sigma-Aldrich), and NLRP3 inhibitor CY-09 (Sigma-Aldrich).
4
Western Blot Analysis of P2X7 and NF-κB
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After being inoculated into a 6-well plate and treated with drugs for 24 h, the cells were disintegrated in a RIPA lysis buffer to extract total proteins, and the lysates were centrifuged at 4°C at a speed of 12000 rpm for 5 min. The supernatant was harvested and preserved at -20°C. After diluting with a sample buffer and heating to 95°C for 10 min, samples containing equal amounts of proteins (20 mg) were separated by SDS-polyacrylamide gel (10%) electrophoresis, using a electrophoresis set-up (Bio-Rad Company, USA), and afterwards transferred onto a PVDF membrane. The chemiluminescent signals were observed using the multifunctional gel imaging system, and the band intensity was quantified in Image-Pro Plus 6.0 software (Media Cybernetics, USA). The primary antibodies included rabbit polyclonal anti-P2X 7 (Abcam, UK; 1:1000 dilution), rabbit polyclonal anti-P65 (CST, USA; 1:1000 dilution), and β-actin (Beijing Zhongshan Biotech Co. 1:800 dilution). The secondary antibody was goat anti-rabbit IgG (Beijing Zhongshan Biotech Co.). Band densities were normalized to each β-actin internal control.
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