The content changes of claudin-5, occludin, E-cadherin, and VE-cadherin proteins were determined in the lung tissues by Western blotting (WB). The right lung was divided into upper, middle, and lower lobes, according to the anatomical structure. The average value of the upper, middle, and lower lobes of lung was taken to compare the protein content in the whole lung between the groups. Lung tissues were homogenized, and 30 g of protein was electrophoresed. Membranes were exposed overnight at 4°C to the E-cadherin antibody (1:1,000; 610181; BD Transduction Laboratories™), VE-cadherin antibody (1:1,000; ab33168; Abcam), occludin antibody (1:1,000; DF7504; Affinity), and claudin-5 antibody (1:500; AF5216; Affinity). Anti-GAPDH antibody (1:1,000; BX008; BioX) was used as the loading control. Immunohistochemistry staining of E-cadherin and VE-cadherin was performed according to the manufacturer’s recommendations.
Af5216
Manufactured by Affinity Biosciences
Sourced in United States
The AF5216 is a laboratory equipment product from Affinity Biosciences. It is a specialized device designed for conducting scientific research and analysis. The core function of the AF5216 is to perform specific tasks within the laboratory setting, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Df7504, by Affinity Biosciences (3 mentions)
Ab33168, by Abcam (1 mentions)
Ab133504, by Abcam (1 mentions)
Af5145, by Affinity Biosciences (1 mentions)
0.45 μm pvdf membrane, by Merck Group (1 mentions)
Sa00001 1, by Proteintech (1 mentions)
Sa00001 2, by Proteintech (1 mentions)
Gb11132, by Wuhan Servicebio Technology (1 mentions)
Gb12001, by Wuhan Servicebio Technology (1 mentions)
Gb12002, by Wuhan Servicebio Technology (1 mentions)
Gb11188, by Wuhan Servicebio Technology (1 mentions)
Af5103, by Affinity Biosciences (1 mentions)
Horseradish peroxidase conjugated goat anti mouse rabbit igg secondary antibody, by Cell Signaling Technology (1 mentions)
Tanon 5200, by Shanghai Tanon Science & Technology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Ab115730, by Abcam (1 mentions)
Supersignal west atto ultimate sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Bca protein assay kit, by Ipsen (1 mentions)
Ripa lysis buffer, by Ipsen (1 mentions)
A4867, by ABclonal (1 mentions)
6 protocols using af5216
1
Lung Tight Junction Protein Quantification
2
Western Blot Analysis of Oxidative Stress
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Tissue and cell lysates were separated, transferred to 0.45 μm PVDF membranes (Millipore, America), blocked and incubated in primary antibodies (metallothionein 2, MT2, 1 : 200, Immunoclone, America, ICA868Hu01). BCL2(1 : 1000, 15071), cleaved-caspase 3 (1 : 1000, 9661S), SOD2(1 : 1000, 13194), CAT (1 : 1000, 12980) (CST, America). cytochrome C (1 : 500, Abcam, Britain, ab133504). Bax (1 : 1000, 60267-1-Ig), VDAC1(1 : 1000, 66345-1-Ig) (Proteintech, Wuhan, China), malondialdehyde (MDA) (1 : 1000, Novus, America, NBP2-59366). claudin 5 (1 : 1000, #AF5216), ZO1 (1 : 1000, #AF5145), occludin (1 : 1000, #DF7504) (Affinity, Jiangsu, China), and β-actin (1 : 800, Boster, Wuhan, China, BA2305), and followed by incubation in HRP-conjugated secondary antibodies (Proteintech, Wuhan, China, SA00001-1 and SA00001-2). The membranes were visualized by the ECL system (Thermo, America, 32132), and the quantification of the band intensity were calculated by the ImageJ software [14 , 15 (link)].
3
Rat model of blood-brain barrier dysfunction
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JFG (0012007009) was bought from Shandong New Times Pharmaceutical Co., Ltd. Primary antibodies of anti-β-actin (GB12001, Servicebio, Wuhan, China), anti-GAPDH (GB12002, Servicebio, Wuhan, China), anti-MMP9 (GB11132, Servicebio, Wuhan, China), anti-PI3K (GB11525, Servicebio, Wuhan, China), anti-AKT (GB13427, Servicebio, Wuhan, China), anti-p-AKT (AF0016, Affinity Biosciences, USA), anti-IL-1β (AF5103, Affinity Biosciences, USA), anti-Occludin (DF7504, Affinity Biosciences, USA), anti-Claudin-5 (AF5216, Affinity Biosciences, USA), and anti-TNF-α (GB11188, Servicebio, Wuhan, China). Evans Blue (CAS: 314-13-6) was purchased from Bioengineering (Shanghai) Co., Ltd. (Shanghai, China). The Collagenase IV (C8160) and rabbit fluorescein conjugated antibody were purchased from Servicebio (Wuhan, China). Adult male rats (Sprague-Dawley rat, 300 ± 20 g) were purchased from Pengyue Laboratory Animal Breeding Co., Ltd. (Jinan, Shandong, China).
4
Immunoblotting of Cell Signaling Proteins
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Lysates were harvested from cells in RIPA lysis buffer (P0013B, Beyotime) containing a protease inhibitor cocktail. Equal amounts of protein were separated via sodium dodecyl sulphate‐polyacrylamide gel electrophoresis, and then transferred to polyvinylidene fluoride membranes electrophoretically. After incubation with blocking buffer, the membrane was incubated with antibodies against iNOS (1:1000, 18985‐1‐AP, Proteintech group), GFAP (1:5000, 60190‐1‐Ig, Proteintech group), ZO‐1 (1:1000, 21773‐1‐AP, Proteintech group), Occludin (1:1000, 27260‐1‐AP, Proteintech group), Claudin‐5 (1:1000, AF5216, Affinity), TAF1 (1:1000, 20260‐1‐AP, Proteintech group), Histone‐H3 (1:1000, 17168‐1‐AP, Proteintech group), or GADPH (1:3000, 60004‐1‐AP, Proteintech group) overnight at 4°C. After three washes, the membrane was incubated with a horseradish peroxidase‐conjugated goat anti‐mouse/rabbit IgG secondary antibody (1:2000, 7076P2/7074P2, Cell Signaling). Signals were detected by Automatic Chemiluminescence Image Analysis System (Tanon 5200, Tanon Science & Technology). Quantification of the individual protein bands was performed via densitometry using Image J software.
5
Western Blot Analysis of Cell Signaling
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Cells were lysed in RIPA lysis buffer (Epizyme, PC101) supplemented with a protease/phosphatase inhibitor cocktail. Proteins were quantified by BCA Protein Assay Kit (Epizyme, ZJ101), supplemented with the loading buffer, and placed in a metal bath at 95 °C for 10 min. After being separated by SDS–polyacrylamide gel electrophoresis, proteins were transferred to PVDF membranes and blocked in PBS containing 5% BSA and 0.1% Tween20. Then, membranes were incubated with primary antibodies (DR6 (1:100, rabbit, bs-7678R, Bioss), Cldn-5 (1:1000, rabbit, AF5216, Affinity), Glut-1 (1:1000, rabbit, ab115730, Abcam), active β-catenin (1:1000, rabbit, 8814, CST), total β-catenin (1:1000, rabbit, 8480, CST), Phospho-JNK (1:1000, rabbit, AP1337, Abclonal), JNK (1:1000, rabbit, A4867, Abclonal) and HRP-Conjugated GAPDH (1:10,000, HRP-60004, Proteintech)) overnight at 4 °C, followed by corresponding secondary antibodies, and detected with SuperSignal West Atto Ultimate Sensitivity Substrate (Thermo Fisher Scientific, A38555) by ChemiDoc MP Imaging System (Bio-Rad, USA). Fiji software was used to quantify band densities. The quantification of phosphorylated proteins was normalised to their total proteins. GAPDH was used as the housekeeping protein, and the data were presented as fold change to the control group.
6
Immunohistochemical Analysis of AQP4 and Claudin-5
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After embedding in paraffin wax, the tissues were sliced to 4 μm thickness, then incubated in 0.3% H2O2 for 30 minutes and incubated in 0.1% Triton X-100 for 20 minutes. Next, the sections were incubated with primary antibodies, including anti-Aquaporin-4 (AQP4) antibody (1 : 50; AF5164; Affinity) and anti-claudin-5 antibody (1 : 50; AF5216; Affinity) overnight at 4°C and secondary antibody for 60 min at 37°C. Lastly, the sections were stained with DAB to develop the color and counterstained with hematoxylin.
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