The protein of tissues and cells were extracted via RIPA lysis (Beyotime). The concentration of protein was measured using BCA kits (Beyotime). Following, the protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and were transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking, the membranes were incubated with the primary antibodies (ADAM8, 1:1,000, Proteintech; α-SMA, 1:1,000, Affinity; FSP1, 1:1,000, Affinity; VE-cadherin, 1:1,000, ABclonal, Wuhan, China; TGF-β1, 1:1,000, ABclonal; Smad2, 1:1,000, Affinity; p-Smad2, 1:1,000, Affinity; Smad3, 1:1,000, ABclonal; p-Smad3, 1:1,000, ABclonal) at 4°C overnight. The membrane was washed using PBS following incubation with corresponding secondary antibodies. The image was analyzed by Gel-Pro Analyzer (Media Cybernetics, Bethesda, MD, USA).
Ve cadherin
Manufactured by ABclonal
Sourced in China
VE-cadherin is a transmembrane glycoprotein that plays a crucial role in the formation and maintenance of adherens junctions between endothelial cells. It is essential for the regulation of vascular permeability and the integrity of the endothelial barrier.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Beyotime (1 mentions)
Tgf β1, by ABclonal (1 mentions)
Ripa lysis, by Beyotime (1 mentions)
Adam8, by Proteintech (1 mentions)
Smad2, by ABclonal (1 mentions)
P smad2, by ABclonal (1 mentions)
P smad3, by ABclonal (1 mentions)
Smad3, by ABclonal (1 mentions)
Gel pro analyzer, by Media Cybernetics (1 mentions)
α sma, by Proteintech (1 mentions)
Cy3 labeled goat anti rabbit igg, by Abcam (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Triton x 100, by Beyotime (1 mentions)
Dmem f12 medium, by Biosharp (1 mentions)
Keap1, by Proteintech (1 mentions)
Caspase 1, by GeneTex (1 mentions)
Twist, by Proteintech (1 mentions)
Snail, by Proteintech (1 mentions)
α sma, by Boster Bio (1 mentions)
Nlrp3, by ABclonal (1 mentions)
3 protocols using ve cadherin
1
Protein Extraction and Western Blot Analysis
2
Isolation and Characterization of PMVECs
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The collected lung tissues were cut into small pieces and grinded to obtain cell suspension. Subsequently, CD45− and CD31+ PMVECs selected by the CD45 and CD31 magnetic beads (Miltenyi Biotec, Gladbach, Germany; 10 μL beads/107 cells) were inoculated onto Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) medium (Biosharp, Hefei, China) supplemented with 10% fetal bovine serum (FBS) in 5% CO2 at 37℃. Immunofluorescence was used to detect the expression of CD31 and vascular endothelial cadherin (VE-cadherin) in the PMVECs. Briefly, the PMVECs were fixed in 4% paraformaldehyde (Sinoreagent, Shanghai, China) and permeabilized in 0.1% Triton X-100 (Beyotime Biotech, Shanghai, China). The PMVECs were incubated with anti-rabbit CD31 (1:100; Abclonal, Shanghai, China) and VE-cadherin (Abclonal, Shanghai, China) at 4℃ overnight. Then, the cells were incubated with Cy3-labeled goat anti-rabbit IgG (1:1000; Abcam, Cambridge, UK) at room temperature for one hour. 2-(4-amidinophenyl) 6-indolecarbamidine dihydrochloride (DAPI; Aladdin, Shanghai, China) was used for counterstaining nuclei. The images were captured using a fluorescence microscope (Olympus, Japan).
3
Western Blot Analysis of Cardiac Markers
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Total protein samples were extracted from tissues or cells using the same procedure previously described [36 (link)]. Briefly, heart tissues or cardiomyocytes were lysed by RIPA buffer. After centrifugation, the supernatant was collected. Protein extracts were mixed with SDS‐PAGE. After electrophoresis, the protein transferred onto a nitrocellulose membrane. Then, the membranes were blocked and incubated with the primary antibodies: BNP (1:1000, ABclonal, China), β-MHC (1:1000, ABclonal, China), Caspase-1 (1:1000, GeneTex, USA), Nrf2 (1:1000, Proteintech, China)，HO-1 (1:1000, Wanleibio, China)，NLRP3 (1:1000, ABclonal, China)，Gasdermin-D (ABclonal, China), USP14 (1:1000, Proteintech, China), Snail (1:1000, Proteintech, China)，Twist (1:1000, Proteintech, China)，Slug (1:1000, ABclonal, China), CD31 (1:1000, ABclonal, China), VE-cadherin (1:1000, ABclonal, China), α-SMA (1:1000, BOSTER, China), Keap1 (1:1000, Proteintech, China), type I collagen (1:1000, ABclonal, China), type Ⅲ collagen (1:1000, affinity, USA).
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