71-1500

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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of PC-3 (Lane 1), MDCK (lane 2), and U-2 OS (Lane 3). The blots were probed with Anti-Occludin Rabbit polyclonal Antibody (Product # 71-1500, 2 µg/mL) and detected by chemiluminescence Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP conjugate (Product # G-21234, 1:5000 dilution). Isoforms of 23kDa and 53kDa bands corresponding to Occludin was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0341BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

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Experimental details: Western blot analysis of MDCK cells using Rb anti-Occludin (Product # 71-1500).

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Experimental details: Knockout of Occludin was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR777079_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of Occludin was performed by loading 30 µg of Caco-2 Cas9 (Lane 1) andCaco-2 Occludin KO (Lane 2) membrane enriched extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-Occludin Polyclonal Antibody (Product # 71-1500, 1.5 µg/mL dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:10,000 dilution) using the iBright™ FL 1500 (Product # A44115). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to Occludin. Uncharacterized bands were observed in all the samples between ~30-50 kDa.

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Experimental details: Western blot was performed using Anti-Occludin Polyclonal Antibody (Product # 71-1500) and 58 kDa, 52 kDa and 31 kDa bands corresponding to Occludin was observed across in MCF7 and HEK-293 and not in HeLa and BJ which are reported to be low expressing for the protein. An uncharacterized band ~60 kDa was also observed. Whole cell extracts (40 µg lysate) of HEK-293 (Lane 1), HeLa (Lane 2), BJ (Lane 3) and MCF7 (Lane 4) were electrophoresed using NuPAGE™ 10% Bis-Tris Protein Gel (Product # NP0301BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1.5 µg/mL dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Reagent Kit (Product # WP20005).

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Experimental details: Immunofluorescent analysis of Occludin using a polyclonal antibody (Product # 71-1500).

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Experimental details: MDCK canine cells stained with Rabbit anti-Occludin (Product # 71-1500) and Zymed's FITC-Goat Anti-Rabbit IgG (H+L) (Product # 62-6111).

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Experimental details: Immunofluorescent analysis of Occludin using a polyclonal antibody (Product # 71-1500).

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Experimental details: Immunofluorescence analysis of Occludin was performed using 90% confluent log phase Caco-2 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with Occludin Rabbit Polyclonal Antibody (Product # 71-1500) at 2 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cell junctional localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.

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Experimental details: Figure 3 Representative confocal photomicrographs comparing localization patterns of claudins-1 (C1), -2(C2), -4(C4) and -7(C7) and occludin (Ocld) in Misoprostol treated (M+, right panel) and untreated (M-, left panel) cervix . The FIT-c (green) and Cyanine-3 (Cy-3-red) labelled secondary antibodies were used; DAPI (blue) for nuclear staining with overlays of red, green and blue (RGB) channels. In the untreated cervix, occludin (red) shifted to intermediate and more superficial layers (3B-red). In the untreated cervix, occludin (3A-red) and claudin-2 (3A green) were expressed in basal layers only (3A-overaly) but following Misoprostol treatment, de novo expression of claudin-2 (3B-green) in the intermediate layer resulted in both now localizing in the basal and intermediate layers (3B-overlay). Compared to diffuse lattice pattern in basal to intermediate layers in the untreated cervix (3C red), Misoprostol induced expression of claudin-7(3D red) in basal and intermediate layers. Claudin-4 was expressed mainly in the nuclei throughout the ectothelium (3C green) in the untreated cervix, with de novo cytoplasmic expression in Misoprostol-treated tissue (3D & 3F green). Claudins 1 and 4 exhibit nuclear and plasma membrane expressions in Misoprostol-treated cohort (3F). Arrows at basement membranes; S-stroma, E-epithelium. 3A- 2 x 2 tile; x 20air objective. NA-0.8, Bar = 8 mum.

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Experimental details: FIGURE 6: Rab25 depletion alters the transepithelial resistance and claudin expression. (A) TER was measured in all cell lines from day 3 to day 15 on Transwell filters. During this period, the media were changed every day, and TER was measured in 24-well Transwell inserts. Results are expressed in ohm-cm 2 and are representative of three separate experiments. (B) Equal amounts of total protein lysates (20 mug) from Control, Rab25KD, and Rescue cells were analyzed by Western blots for tight junction proteins. The blots were subsequently stripped and reprobed for beta-actin as a loading control. The results are representative of three separate experiments.

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Experimental details: Figure 6 Expression of TJ proteins in transmigrating cells favors their passage across RBMECs. A) Analysis by Western blots of the expression of occludin, claudins 1 to 5 and CRTAM in NIH-3T3and L-fibroblasts, ReNcells CX and RBMECs. B) The transmigration of NIH-3T3 and L-fibroblasts acrossRBMECs is independent of the CM present in the basal compartment, but is significantly higher inNIH-3T3 fibroblasts that express claudin-2 than in L-fibroblast that do not express TJ proteins.N = 3, F(1,6) = 232.22, ***P

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Experimental details: Figure 3 The expression of tight junction mRNA and proteins in Caco-2 EP2 receptor transfectants. (A) The mRNA expression for tight junction proteins (ZO-1, occludin, claudin-1, claudin-2 and claudin-4) in Caco-2 EP2S and EP2A cells was measured by Q-PCR. The expression of gene of interest (GOI) was normalized with the internal control (Actin) and expressed as fold changes with that of the vector control. (B) A representative immunoblot showing constitutive expression of the important tight junction proteins (ZO-1, occludin, claudin-1, claudin-2 and claudin-4) in EP2S, EP2A and vector control cells. Actin used as internal control. (C) Densitometry analysis for claudin-4 expression in EP2S, EP2A and vector control cells. EP2A compared statistically with the vector control. p

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Experimental details: Figure 5 Effects of suppression of cofilin activation or overexpresssion of TJ proteins on the capsaicin-induced cofilin dephosphorylation and decrease in occludin expression. (A-C) Flag-HA tagged cofilin and LIMK, and EGFP-occludin and claudin-1 stable transfectants were established. Monolayers prepared from the transfectants and non-transfected MDCK cells were exposed to 300 uM capsaicin for the time indicated. After lysis, protein expression was analyzed by immunoblotting. Each experiment was performed with at least two different clones and repeated at least twice. (D) MDCK monolayers were pretreated with vehicle or 50 uM TFP for 30 min, and then exposed to 100 uM capsaicin for the time indicated. The levels of occludin and phospho-cofilin were analyzed by western blotting.

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Experimental details: Figure 7 Cofilin activation, decrease in occludin expression and actin alteration in the recovery phase of capsaicin treatment. (A) Western blot detection of phospho-cofilin and occludin in total extracts exposed to 300 uM capsaicin for the times indicated. (B) MDCK monolayers were exposed to 300 uM capsaicin for the time indicated, and then fixed and stained with rhodamine-phalloidin to detect F-actin. Images from each z-section were deconvoluted and overlayed. Bar: 10 um.

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Experimental details: Figure 3 Effects of capsaicin on the distribution of TJ proteins and F-actin. (A, B) Monolayers were exposed to vehicle (A) or 300 uM capsaicin (B) for 45 min and were labeled with each TJ antibody (green), rhodamine-phalloidin (red) and Hoechst (blue). Images were collected as a Z-series, and then deconvoluted and overlayed to display a single composite projection. Top : XY sections of merged images. Bottom : XZ sections of merged images. Scale bar: 10 um. The corresponding TJ antibodies are listed above the images. (C) 3D projection images in a 45deg-angle of claudin-1 and tricellulin staining, plus F-actin. Scale bar: 10 um. (D) MDCK monolayers exposed to vehicle (top) or capsaicin (bottom) as above were stained with E-cadherin. The colors corresponding to each antibody are listed above the images. Scale bar: 2.5 um. (E) Schematic explanation of the protein distributions.

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Experimental details: Figure 1 Experimental workflow and characterization of SW480-ADH Mock and Snail1 cells. (A) Scheme of the workflow followed in this study. (B) Representative phase-contrast micrographs (upper panels) and confocal laser immunofluorescence images showing F-actin staining (lower panels) of SW480-ADH Mock and Snail1 cells. Scale bar, 20 um (upper panels) and 10 um (lower panels). (C) Western blot analysis of whole-cell extracts from SW480-ADH Mock and Snail1 cells showing Snail1 overexpression and its effect on the expression of epithelial (E-cadherin, Occludin and Claudin-7) and mesenchymal (Lef-1) markers. beta-Tubulin was used as a loading control.

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Experimental details: Fig. 4 Endothelial cells were infected for 2 h with S. aureus (MOI 10:1) or left uninfected. As indicated, cells were pretreated for 20 min with amitriptyline (Ami) (20 muM), Tiron (10 mM), or NAC (10 mM) before infection with S. aureus . Immunofluorescence stainings were performed with antibodies against ZO1, ZO2, occludin, or E-cadherin for determination of the degradation of these TJ proteins. The presented pictures are representative of the results of at least three independent experiments. Scale bar is 25 mum

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Experimental details: Figure 2. Induction of epithelial markers by 1 alpha ,25(OH) 2 D 3 in SW480-ADH cells. (A) Analysis by immunofluorescence and confocal laser scanning microscopy of the expression of various adhesion proteins in cells treated with 10 -7 M 1alpha,25(OH) 2 D 3 for the indicated times or left untreated (control): E-cadherin (a-c); beta-catenin (d-f). (B) Same as in A with longer treatments: a-c, occludin; d-f, ZO-2; g-k, vinculin. Vinculin expression was analyzed at two sections: basal (g, i, and k) and apical (h and j). Bars, 10 mum.

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Experimental details: Figure 6 Cx43 mutant-expressing REKs exhibited reduced transepithelial resistance when compared with Cx43 overexpressing REKs in the absence of changes in the expression or localization of junctional proteins ( A ) Transepithelial resistance measurements of confluent monolayer cultures were taken daily for 5 days and the resistance from days 2-4 were averaged and plotted. ( B ) E-cadherin, claudin1 and occludin were localized by immunofluorescent labelling in REKs overexpressing GFP-tagged Cx43, G138R or fs260. Western blot analysis did not reveal any changes in the expression of occludin, E-cadherin or claudin-1 ( C and D ). * P

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Experimental details: Figure 4 Capsaicin decreases occludin protein content and the protein-protein interactions involving occludin in TJ. (A,B) MDCK monolayers were exposed to ethanol control or 300 uM capsaicin for 45 min. (A) The cellular localization of the TJ proteins occludin and claudin-1 was examined by immunofluorescence. Scale bar: 10 um. (B) Western blot detection of occludin, tricellulin, Zo-1, claudin-1, E-cadherin and actin in the cytosol, membrane fractions and total cell extracts. The densitometic analysis of total proteins from three independent experiments performed with NIH ImageJ software. **represents p

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Experimental details: Figure 5 Knockdown of Rai14 in the Sertoli cell epithelium with an established TJ-permeability barrier in vitro by RNAi disrupts actin filament organization and the TJ barrier. (A) Sertoli cells cultured alone on Matrigel-coated 12-well dishes for 2-day with an established TJ-permeability barrier were transfected with Rai14 siRNA duplexes (Rai14 RNAi) versus non-targeting control duplexes (Ctrl RNAi) at 100 nM using Ribojuice transfection medium for 24 hr, thereafter, cells were washed twice and cultured in F12/DMEM for 12 hr to allow recovery. Thereafter, cells were transfected again under the same conditions for another 24 hr. Thereafter, cells were rinsed with fresh F12/DMEM and cultured for an additional 12 hr before termination, and used to prepare lysates for immunoblotting using antibodies against several BTB-associated constituent or regulatory proteins. A knockdown of Rai14 by ~50% was noted in which the control was arbitrarily set at 1 against which statistical comparison was performed (B) without any apparent off-target effects (A). The findings shown herein are the results of 3 independent experiments excluding pilot experiments which were used to establish optimal experimental conditions, such as different concentrations of siRNA duplexes and Ribojuice. It was noted that we achieved only ~50-60% knockdown of Rai14 in several pilot experiments, unlike other target genes [ e.g ., Scribble, beta1-integrin, and P-glycoprotein] wherein we could silence the target gene

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Experimental details: Figure 1 Rai14 is an actin-binding protein in the rat testis. ( A ) A study by RT-PCR to confirm the expression of Rai14 in adult rat testis, Sertoli cells (SC, isolated from 20-day-old rat testes and cultured for 4-day), and germ cells (GC, isolated from adult rat testes and cultured for 16 hr). ( B ) Immunoblotting also confirmed the expression of Rai14 in the rat testis, Sertoli and germ cells, and the relative expression of Rai14 in SC vs. GC was shown in the histogram with n = 3 experiments in which the relative expression level of Rai14 in the testis was arbitrarily set at 1 so that the relative expression level between these samples can be compared. ( C ) The specificity of the anti-Rai14 antibody ( Table 1 ) was assessed by immunoblotting using lysates of GC (20 ug protein). ( D ) Using the specific anti-Rai14 antibody, Rai14 was shown to be an actin-binding protein by co-immunoprecipitation (Co-IP); however, Rai14 did not structurally interact with any of the BTB-associated proteins including several actin-binding and regulatory proteins ( e.g ., Arp3, drebrin E, Eps8) and vimentin (an intermediate filament-based constituent protein). However, Rai14 was found to structurally interact with an actin cross-linking protein palladin which is known to be involved in conferring actin filament bundles in other mammalian cells [48] . ( E ) Rai14 (red) was also shown to be an actin-binding protein by dual-labeled immunofluorescence analysis in which it co-localized with F-acti

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Experimental details: Figure 2 Individual BBB components are affected in the Ngamma1-KO brains. ( a ) Immunohistochemistry shows that astrocytic endfeet marker AQP4 is expressed at high level in Ctr mice, but dramatically reduced in Ngamma1-KO mice. High power confocal Z-projection images show that AQP4 co-localizes with endothelial cell marker CD31 in Ctr but not Ngamma1-KO brains. ( b ) Occludin and claudin-5, but not PDGFRbeta, are significantly decreased in Ngamma1-KO brains. ( c ) Representative Western blot of AQP4, occludin, claudin-5, PDGFRbeta, and actin using striatum lysates from Ctr and Ngamma1-KO mice. Full blots of these proteins are shown in Supplementary Figure 14 a . Western blot was quantified by densitometry. All bands were normalized to actin (n=6). Scale bars represent 100 mum (low magnification images in a and b ) and 20 mum (high magnification images in a). Data are shown as mean +- sd. *p

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Experimental details: Figure 4 Constitutive expression of EspF and Map depletes tight junction proteins. ( A ) Cell lysates derived from untransfected, EGFP, EGFP-Tir, EGFP-EspF and EGFP-Map cell lines were separated by electrophoresis on 12% SDS-polyacrylamide gels and transferred to PVDF membranes. Equal amounts of cell lysates were loaded after normalizing with GAPDH. Blots were probed with the indicated primary antibodies and HRP-conjugated secondary antibodies. Full-length blots are presented in Supplementary Figure S3 . ( B , C ) The x-ray films were scanned and quantitative analysis was performed by measuring band intensities using ImageJ software. The expression of different tight junction proteins in these cell lines was normalized with respect to untransfected cells and fold changes were calculated. Data are represented as means +- s.e.m. from six independent experiments using two biological replicates for all cell lines; **represents p-values < 0.001. ( D ) The cell surface proteins were labeled with membrane impermeable Sulfo-NHS-Biotin and immobilized on streptavidin-agarose beads. The beads were washed and subjected to SDS-PAGE. E-cadherin was used as a loading control for cell surface proteins. Full-length blots are presented in Supplementary Figure S4 . ( E ) The levels of cell surface proteins were estimated by densitometric scanning of x-ray films using ImageJ software. Shown are fold changes with respect to untransfected cells. Error bars represent means +- s.e.m. from three i

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Experimental details: Supplementary Figure 5 Agrin 000 and agrin 748 have no influence on the junctional localization of claudin 5, occludin, and ZO-2 in bEnd5 cells. A-C bEnd5 cells cultured on agrin 000, on agrin 748, or under control conditions were stained for claudin-5 ( A ), occludin ( B ) and ZO-2 ( C ) after 48 h in culture. Representative micrographs from 3-4 independent experiments are shown. Bar 50 mum. D-F The gray values of the junctional IF signal of claudin 5 ( D ), occludin ( E ), and ZO-2 ( F ) were measured with ImageJ software. Symbols represent the mean values of each independent experiment ( D , F , n = 3, E n = 4), and the overall means are represented by the horizontal lines . The gray values were normalized to the control condition for every independent experiment (GIF 196 kb)