Claudin 5 Antibody - #AF5216

Fig. 4. Inhibition of protein palmitoylation ameliorates PA induced Sertoli cell dysfunction. (A) Analysis of the palmitoylation levels of proteins extracted from the testes of mice administered or not administered the PA injection, with or without gavage of 2-BP (n = 6 for Control, n = 7 for PA and 2-BP + PA). (B) Analysis of the palmitoylation levels of proteins extracted from primary Sertoli cells, Leydig cells and germ cells, which were treated with or without 0.4 mM PA (n = 3). (C) Inhibition of palmitoylation by 2-BP suppressed PA-induced ER stress in Sertoli cells. Translational expression levels of ER stress-related genes were analyzed using Western blotting (n = 3). (D) ROS detection using DCFH-DA staining. The fluorescence densities were calculated using ImageJ (n = 3). Scale bar: 50 μm. (E) TER detection of primary Sertoli cell barriers. The cells were incubated with PA (PA), with PA combined with 2-BP (2-BP + PA), or with the vehicle (Control) for 3 days after barriers were formed on day 4 (n = 5). (F) FITC-dextran permeability assessment of primary Sertoli cell barriers. The cells were treated PA (PA), with PA combined with 2-BP (2-BP + PA), or with the vehicle (Control) for 24 h after cell barriers were formed (n = 5). (G)Tight junction protein levels were examined by western blotting in TM4 Sertoli cells (n = 3). The relative intensities of bands in western blotting results were quantified by ImageJ and normalized to β-actin levels. Data are presented as mean ± SD. n. s., no significant difference vs. Control group. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. Control group. #P < 0.05, ##P < 0.01 and ###P < 0.001 vs. PA group.

Figure 1 BBB dysfunction and BEC disruption in post-mortem Alzheimer’s disease brains. (A) Fibrinogen/CD31, Cldn5 and Aβ/Glut1 staining in the cortex and (B) hippocampus of post-mortem health control and patients with Alzheimer’s disease. (C) Quantification of Cldn5 and (D) Glut1 intensity, and (E) Aβ plaques in the cortex and hippocampus of healthy controls and patients with Alzheimer’s disease. Black frames in the low-magnification images indicate the location of the high-magnification images to the right. Arrows indicate Aβ plaques, and arrowheads indicate the fibrinogen leakage and decreased Cldn5 and Glut1 in patients with Alzheimer’s disease. Scale bar = 200 µm in A and B. Data are presented as mean ± SEM, n = 5, *P < 0.05, **P < 0.01.

Fig. 3 Treatment with GTA improved BBB permeability in the peri-infarct area on day 7 after cerebral ischemia. A The representative image of extravagated dextran and IgG in the peri-infarct area. B and C Quantification of dextran and IgG intensity based on A. D The representative image of Evans blue leakage. E Quantification of D. F, Western blotting of tight junction proteins in the peri-infarct area. G–I Quantification of ZO-1, Occludin and Claudin-5 based on F. n = 5 for B and C, n = 4 for E–I. Compared with Sham, *P 

Fig. 5 MenSCs promoted TJ- and bile transport function-related protein expression and reduced fibrosis-related protein expression. A Western blot analysis protein levels of Claudin-1, Claudin-3, Claudin-5, Claudin-7 and Occludin in liver tissue of different groups (n = 3 for each group). B BSEP, OATP2 and NTCP1 expression in liver tissues of different groups (n = 3 for each group). C, D Liver COL1A1, α-SMA, TGF-β1 and β-catenin expression among different groups (n = 3 for each group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Figure 2. P-glycoprotein silence attenuates adhesion molecule expression, myeloid cell accumulation and blood-brain barrier damage in experimental ischemic stroke. Mice were intracerebroventricularly injected with P-glycoprotein (P-gp) or negative control (NC) siRNA (1.5 μL/10 g body weight) 48 h prior to MCAO/R surgery. Twenty-four hours after the surgery, brains were harvested for immunohistochemical and Western-blotting assays. (A) Immunohistochemical localization and quantification of ICAM-1 and VCAM-1 expression (n = 6). (B) Representative immunostaining images and quantification of neutrophiles (MPO) and macrophages (F4/80) (n = 6). (C-E) Representative Western-blotting images and quantification of tight junction proteins (Claudin-5, Occludin, and ZO-1) (n = 3). Scale bars: 40 μm. One-way ANOVA followed by the post hoc Tukey test. All data are mean ± SD, *P

Figure 5. P-glycoprotein silence alleviates endothelial dysfunction following oxygen glucose deprivation/reoxygenation. Endothelial cells (bEnd.3) were transfected with P-glycoprotein (P-gp) or negative control (NC) siRNA, or un-transfected, and then subjected to either oxygen glucose deprivation/reoxygenation (OGD/R) treatment or normal culture conditions. Twenty-four hours thereafter, cells were harvested for immunofluorescence staining, adhesion and transendothelial migration, real-time PCR gene expression and Western-blotting analyses. (A) Representative Western-blotting images and quantification of P-gp levels (n = 3). (B) Quantification of mRNA levels for TNF-α, IL-1β, MMP-2, and MMP-9 via quantitative real-time PCR assay (n = 4). (C) Representative immunofluorescence staining images and quantification of ICAM-1 and VCAM-1 expression (n = 3). (D) Representative images and quantification for fluorochrome-labeled leukocyte adhesion to endothelial cells and transendothelial migration (n = 3). (E) Representative immunofluorescence staining images for tight junction proteins (Claudin-5, Occludin, and ZO-1). (F) Representative immunoblots and quantification for the expressions of Claudin-5, Occludin, and ZO-1 (n = 3). Scale bars, 40 μm. One-way ANOVA followed by the post hoc Tukey test for A, E, and F. Mann-Whitney test for B, C, and D. All data are mean ± SD, *P

Fig. 3. LSP improves TJ expression and inhibits apoptosis in db/db mice. (A) Representative immunohistochemistry images of ZO-1 and Claudin-5 in the mouse retina (magnification, ×40). (B-C) Quantification of the expression of ZO-1 (B) and Claudin-5 (C). (D) Western blotting detection of Bax, Bcl-2, and Caspase-9. (E-F) Bar charts indicate Bax/Bcl-2 (E) and active caspase-9/pro-caspase-9 (F). nsp > 0.05, ***p ≤ 0.001.

Fig. 6 ICS II improved BBB integrity after cerebral I/R by upregulating the expression of tight junction-related proteins. a Representative images of immunoblotting staining of claudin 5, occludin and ZO 1 in the penumbra. b Quantitative analysis of occludin. c Quantitative analysis of claudin 5. d Quantitative analysis of ZO 1. *P < 0.05, **P < 0.01 vs sham; #P < 0.05, ##P < 0.01 vs MCAO; n = 6 per group

Fig. 6| ICS II improved BBB integrity after cerebral I/R by upregulating the expression of tight junction-related proteins. a Representative images of immunoblotting staining of claudin 5, occludin and ZO 1 in the penumbra.