51-6100

antibody from Invitrogen Antibodies

Targeting: CLDN2

Provider product page for 51-6100

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Antibody data

Antibody Data
Antigen structure
References [100]
Comments [0]
Validations
Western blot [1]
Immunocytochemistry [1]
Immunohistochemistry [1]
Other assay [60]

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Product number: 51-6100 - Provider product page
Provider: Invitrogen Antibodies
Product name: Claudin 2 Polyclonal Antibody (MH44)
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: This antibody is specific for the ~ 22 kDa Claudin-2 protein and does not appear to cross-react with related endogenous proteins. Reactivity was confirmed by western blotting with rat kidney and by immunostaining of rat small intestine. This antibody is confirmed reactive with human, rat, and dog Claudin-2. The reactivity of this antibody with other species has not been determined.
Reactivity: Human, Rat, Canine
Host: Rabbit
Isotype: IgG
Antibody clone number: MH44
Vial size: 100 µg
Concentration: 0.25 mg/mL
Storage: -20°C

CLDN2 protein structure - 51-6100 shown in red.

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Experimental details: FIGURE 10: Overexpression of either GFP-Rab11-FIP2-WT or GFP-Rab11-FIP2 (S227E) altered the claudin content of tight junctions. GFP-Rab11-FIP2-WT-on and GFP-Rab11-FIP2(S227E)-on and -off were grown for 5 d postconfluence on Transwell filters. The cells were fixed then stained for claudin-1 (A), claudin-2 (B), or claudin-4 (C) and ZO-1. In the merged image, GFP-Rab11-FIP2s are pseudocolored green, the claudins are red, and ZO-1 is blue. (D) Cells were grown as described, then lysed, run on Western blots, and probed for the different claudins and VDAC. Three separate blots were scanned and quantified using ImageJ and normalized to control (VDAC). *p

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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 GC-C is essential to maintain intestinal barrier function during C. rodentium infection. A : GC-C +/+ and GC-C -/- mice were given a FITC-dextran tracer by oral gavage on day 4 p.i. and serum fluorescence was measured four hours later. Relative to naive mice as well as infected GC-C +/+ animals, GC-C -/- mice have a substantial and significant increase in tracer movement from the gut lumen into the blood indicating a loss of intestinal barrier function. (* P

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Experimental details: FIG 2 Validation of RNA-protein interactions by RNA immunoprecipitation. Huh7 cells were infected with DENV2 NGC at an MOI of 1. Infected cell lysates were incubated with an isotype control antibody or an antibody directed against the indicated cellular proteins. Bound material was captured on Dynabeads protein G for analysis. (A) Western blot analysis of immunoprecipitated material. Input represents 10% of total input sample. IgG lanes represent 20% of immunoprecipitated protein. (B) RT-qPCR analysis of immunoprecipitated RNA. Statistical significance relative to DENV measured by Student's t test: *, P

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Experimental details: Figure 2 Claudin-2 expression is derepressed in the jejunum of Gata4 mutant mice. ( a ) Total RNA was isolated from the jejunum of control and Gata4 DeltaIEC mice (n = 3-4 per group) and RT-qPCR was performed to quantify claudin-2 gene transcripts. **** P < 0.0001 (Student-t test). ( b ) Western blot analysis was performed using a claudin-2 polyclonal antibody on total lysates prepared from control and Gata4 DeltaIEC mice. An actin polyclonal antibody was used as a loading control to monitor protein integrity. Cropped blots are displayed and full-length blots are included in the supplementary information. ( c ) Immunofluorescence detection of claudin-2 on jejunum sections prepared from control and Gata4 DeltaIEC mice. Arrows display typical labeling in the tight junctions. Original magnification: 10X. ( d ) Schematic representation of the Cldn2 gene with its predicted binding sites for GATA. ( e - g ) ChIP analysis of three paired biological samples (n = 3) obtained from the jejunum of control and Gata4 DeltaIEC mice. Data were obtained by qPCR and are expressed as the percent of total DNA input used for precipitation with an antibody against Gata4 relative to the DNA precipitated with normal goat-IgG. * P < 0.05 (ANOVA test). ( h , i ) ChIP analysis from the jejunum of control and Gata4 DeltaIEC mice. Data were obtained by qPCR and are expressed as the percent of total DNA input used for precipitation with an antibody against H3K4me3 ( h ) or H3K27me3 ( i ) relative to the D

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Experimental details: Figure 5 PP242 alleviated the change of claudin-2 expression and intestinal epithelial tight junction function in TNF-alpha treated Caco-2 cells. ( A ) Western blot analysis of claudin-2 after TNF-alpha (10 ng/mL, 48 h) and/or PP242 (1 mum, 24 h) treatment. ( B ) Immunofluorescence staining of claudin-2 after TNF-alpha and/or PP242 treatment in Caco-2 cell monolayers. The green and blue fluorescence were represented the claudin-2 protein and nucleus, respectively. Scale bar: 20 mum. ( C ) The effect of PP242 (1 mum, 24 h) on TER of Caco-2 cell monolayers treated with TNF-alpha (10 ng/mL, 48 h). ( D ) The effect of PP242 on the permeability of FITC-dextran in TNF-alpha treated Caco-2 cell monolayers. Data were shown as mean +- SD and replicated three times. ** p < 0.01, *** p < 0.001, **** p < 0.0001 versus control group. ## p < 0.01, ### p < 0.001 versus TNF-alpha group. n.s., no significant. Ctr, control group.

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Experimental details: Fig. 8 Immunohistochemical detection of claudins in choroid plexuses of developing human brain. Immunoreactivity of Cld-1, -2, and -3 is presented on the left, central, and right panels , respectively, with hematoxylin counterstaining. a Staining of a WD8 fetal brain. Cld-1 labeling, observed in both LVCP and 4VCP, was continuous and localized apically at intercellular junctions of the epithelium ( arrows in inserted high magnification micrograph ). Cld-2 immunoreactivity displayed a similar apical, but patchy pattern in both LVCP and 4VCP. The higher magnification ( insert in LVCP) illustrates the discontinuous labeling ( arrows ). Cld-3 was also detected in the epithelium of both CP ( arrows ). Inserts are higher magnifications highlighting the continuous staining pattern. b Staining of a WD22 fetal brain. Cld-1 immunoreactivity was present at all epithelial junctions (shown in 4VCP). A continuous apical labeling of the epithelium was observed for Cld-2 (LVCP is shown). Cld-3 labeling was faint but continuous in LVCP epithelial cells. c Staining of a WD39 fetal brain. A distinct and continuous labeling of the choroidal epithelium was observed for all Cld-1 (shown for LVCP), -2 (shown for 4VCP), and -3. The continuous signal for Cld-3 was only associated with the epithelium ( arrows ) and not with stromal vessels ( arrowheads in LVCP). The insert shows a similar Cld-3 staining for 4VCP. Abbreviations as in Fig. 2 . Scale bar 50 mum

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Experimental details: Fig. 4 Differential expression of claudin-2 in choroid plexus during rat brain development. a Representative Western blot of LVCP and 4VCP dissected from E19, P2, and adult animals (10 mug per lane). Cld-2 expression increased in CPs during development, and was distinctively higher in LVCP than in 4VCP of E19 and P2 animals ( upper panel band at 22 kDa). Actin used as a loading indicator is shown in the lower panel (42 kDa band). b - e Cld-2 immunoreactivity ( red ) in the developing and adult brain. LVCP and 4VCP sections have been labeled simultaneously and pictures generated with identical camera settings. Cld-2 staining was associated with the epithelium throughout the LVCP of E19 animals ( arrow ), while the staining in 4VCP was faint and patchy, with large portions of the epithelium remaining unlabeled ( b ). In P2 animals ( c ), the staining was more homogeneous in 4VCP and became continuous between epithelial cells ( insert ), as in the LVCP ( right panel ). The epithelial staining was strong in all CPs at P9 ( d arrow ) and in adult animals ( e ). In b , d , e double immunolabeling with anti-RECA-1 Ab allowed visualizing the choroidal vessels unlabeled by the anti-Cld-2 Ab ( arrowheads ). DAPI was used for nuclei staining. Scale bars 20 mum. Abbreviations as in Fig. 2

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Experimental details: Figure 1 Change of claudin-2, LC3B-II, and P62 protein in TNF-alpha treated Caco-2 cell monolayers. ( A ) Western blotting analysis of caludin-2 in Caco-2 cell monolayer after TNF-alpha (10 ng/mL) administration or 3-MA (5 mM) treatment for 48 h. ( B , C ) Western blotting analysis of LC3B-II and P62 in Caco-2 cells after TNF-alpha (10 ng/mL) administration for 48 h. beta-actin was used as an internal control. Data were shown as mean +- SD and replicated three times. ** p < 0.01 versus control group. Ctr, control group.

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Experimental details: Figure 3 Representative photographs of claudin-2, claudin-3, claudin-4, and zonula occludens-1 immunostained in longitudinal jejunal sections of the three experimental groups. Claudin-2 immunoreaction in the sham-operated group (a) was low and was mainly found in the cytoplasm of the intestinal epithelial cells. Claudin-2 expression in the groups exposed to ischemia-reperfusion was substantially higher and was principally found in the lateral membrane of the epithelial cells (b, c). The sham-operated group presented strong membrane claudin-3 immunostaining (d), which was remarkably decreased in the ischemia-reperfusion- (IR-) group (e), but not in the postconditioned- (PC-) group (f). Minimal claudin-4 immunostaining was observed in the jejunal samples of the sham-operated animals (g). The two groups challenged with 60 minutes of mesenteric ischemia and 6 hours of reperfusion (h, i) showed high membrane claudin-4 expression, which was limited to the tips of the villi. Zonula occludens-1 was strongly immunostained in the jejunal samples of the sham-operated animals (j). Following small intestinal ischemia-reperfusion, intestinal epithelial cell with zonula occludens-1 membrane immunostaining notably decreased in the IR-group (k); in the PC-group minimal loss of zonula occludens-1 membrane immunostaining was detected (l). Each photograph was taken with the same magnification. Scale bar: 20 mu m.

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Experimental details: Figure 3 Western blot and RT-qPCR. ( A ) Expression levels of barrier related proteins by densitometric analysis of Western blot (WB). Values are expressed as % of a mean of all controls (ctrls). Bars indicate mean +- standard error of the mean. Asterisk indicates statistically significant difference between groups (* p < 0.05). ( B ) mRNA levels by RT-qPCR. Values are expressed relative to a mean of all controls. Three controls (18%, 3/17) presented a different baseline of housekeeping genes, while two controls (12%, 2/17) presented outliers, which were removed, leaving the final number of controls for mRNA analysis at 12 (70% 12/17). One outlier (3%, 1/33) was removed from the UC group. n = number of UC observations: COX-1 ( n = 16), COX-2 ( n = 11), claudin-2 ( n = 23), claudin-4 ( n = 33), occludin ( n = 31), tricellulin ( n = 29), and DRA ( n = 33). Bars indicate mean +- standard error of the mean. Asterisks indicate statistically significant difference between two groups (* p < 0.05, ** p < 0.01).

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Experimental details: Figure 3 Western blotting analysis. Representative western blotting images of (A) claudin-2, occludin, ZO-1, F-actin and Cdc42 in the intestinal epithelium. Relative expression of (B) Cdc42, (C) occludin, (D) F-actin, (E) ZO-1 and (F) claudin-2 determined by optical densitometry. Results are expressed as the mean +- standard deviation (n=6 randomly selected mice in each group). * P

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Experimental details: Figure 6 The murine jejunal tight junctional network is affected by DXR exposure. ( A ) RT-qPCR of tight junctional genes in jejunal crypt epithelium isolated from control tissues and tissues collected 24 h after DXR from conventionally raised mice ( n = 3/group). All data is presented as fold change normalized to Actb . Student's t test; *p < 0.05, **p < 0.01. ( B ) Representative images of immunofluorescent staining of tight junction associated proteins Claudin-2 (Cldn2), Claudin-4 (Cldn4), and Zona Occludens-1 (ZO-1) in control tissues (Cntrl) and tissues collected 24 h after DXR (24 h) from conventionally raised mice. Images are representative of n = 3 per time point. Scale bar 20 mum.

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Experimental details: Figure 1 Effects of anthocyanins on the protein levels of CLDN1 and CLDN2 in A549 cells. Cells were incubated with 0, 1, 10, or 50 uM C3G ( A ), P3G ( B ), or cyanidin ( C ) for 24 h. Cell lysates were immunoblotted with anti-CLDN1, anti-CLDN2, and anti-beta-actin antibodies. The protein levels of CLDN1 and CLDN2 are represented as a percentage relative to 0 uM. n = 3-4. ** p < 0.01 and NS p > 0.05 compared with 0 uM.

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Experimental details: Figure 2 Effects of anthocyanins on the cellular localization of CLDN1 and CLDN2. Cells cultured on cover glasses were incubated in the absence (control) and presence of 50 uM C3G or P3G for 24 h. ( A ) The cells were stained with anti-CLDN1 (green), anti-ZO-1 (red), and nuclear marker DAPI (blue). ( B ) The cells were stained with anti-CLDN2 (red), anti-ZO-1 (green), and DAPI (blue). Merged images are shown in the right panel. The scale bar represents 10 um. The TJ localizations of CLDN1 and CLDN2 are shown as percentage of control ( n = 132-220 cells). ** p < 0.01 and NS p > 0.05 compared with control.

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Experimental details: Figure 5 Decrease in the protein stability and reporter activity of CLDN2 by C3G. ( A ) Cells were incubated with 10 uM CHX in the absence and presence of 50 uM C3G for 0, 2, 4, and 6 h. Cell lysates were immunoblotted with anti-CLDN2 and anti-beta-actin antibodies. The protein levels of CLDN2 are represented as a percentage relative to control. ( B ) Cells were incubated in the absence (control) and presence of 50 uM anthocyanins for 6 h, and then the reporter activity was measured by the Dual-Luciferase Reporter Assay System. The reporter activity is represented as a percentage relative to control. ( C ) Cells were incubated with 1 µg/mL actinomycin D in the absence and presence of 50 uM C3G for 0, 3, and 6 h. The mRNA level of CLDN2 was measured by quantitative real-time PCR and represented as a percentage relative to 0 h. n = 3-5. ** p < 0.01, * p < 0.05 and NS p > 0.05 compared with control or 0 h.

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Experimental details: Figure 6 Effect of C3G on the degradation and endocytosis of the CLDN2 protein. ( A ) Cells were incubated in the absence (control) and presence of 50 uM C3G, 5 uM MDC, or 10 uM MbetaCD for 24 h. ( B ) Cells were incubated in the absence (control) and presence of 50 uM C3G, 20 uM CHL, or 5 uM MG132 for 24 h. ( C ) Cells were incubated in the absence (control) and presence of 50 uM C3G for 24 h. CAN (0.5 uM) was added into the medium 9 h before collection. Cell lysates were immunoblotted with anti-CLDN2 and anti-beta-actin antibodies. The protein levels of CLDN2 are represented as a percentage relative to control. n = 3-5. ** p < 0.01 compared with control. ## p < 0.01, and NS p > 0.05 compared with vehicle.

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Experimental details: Figure 7 Effect of C3G on the levels of p-p38 and CLDN2. ( A ) Cells were incubated with 10 uM CHX in the absence (control) and presence of 10 uM LY-294002 for 0, 2, 4, and 6 h. Cell lysates were immunoblotted with anti-CLDN2 and anti-beta-actin antibodies. The protein levels of CLDN2 are represented as a percentage relative to 0 h. ( B ) Cells were incubated in the absence and presence of 50 uM C3G for 1 h. Cell lysates were immunoblotted with anti-p-p38 and anti-p38 antibodies. The p-p38 levels are represented as a percentage relative to control. ( C ) Cells were incubated in the absence and presence of 50 uM C3G and 10 uM SB203580 (SB) for 24 h. Cell lysates were immunoblotted with anti-CLDN2 and anti-beta-actin antibodies. The protein levels are represented as a percentage relative to control. ( D ) Cells were incubated with 0, 5, and 10 uM anisomycin for 24 h. Cell lysates were immunoblotted with anti-CLDN2 and anti-beta-actin antibodies. The protein levels are represented as a percentage relative to 0 uM. n = 3-4. ** p < 0.01 and NS p > 0.05 compared with control. ## p < 0.01 compared with vehicle.

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Experimental details: Figure 8 Effects of anisomycin and SB203580 on CLDN2 expression. ( A ) Cells were incubated in the absence (vehicle) and presence of 10 uM SB203580 (SB) for 24 h. Cell lysates were immunoblotted with anti-CLDN2 and anti-beta-actin antibodies. The protein levels of CLDN2 are represented as a percentage relative to vehicle. ( B ) Cells were incubated with 10 uM CHX in the absence (control) and presence of 10 uM SB203580 (SB) for 0, 2, 4, and 6 h. The protein levels are represented relative to 0 h. ( C ) Cells were incubated in the absence (control) and presence of 10 uM C3G and 10 uM SB203580 for 6 h. The mRNA levels are represented as a percentage relative to control. n = 3-4. ** p < 0.01 and * p < 0.05 compared with 0 uM or control. NS p > 0.05 compared with vehicle.

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Experimental details: 3 FIGURE Elevated levels of Claudin-2 in colon of CST-KO mice and elongated epithelial junctions in IBD patients. (A) Western blots and (B) densitometric analyses of Claudin-1 (B), Claudin-2 (C), Occludin (D), Tjp1 (E), Catenin 1 (F) and Cadherin 1 (G). H and I, TEM micrographs (n = 3) showing tight junction (TJ), adherens junction (AJ) and desmosomes (Des) in healthy control and IBD patients. Microvillus (MV). J-L, Morphometrical analyses (n = 20-54) showing increased lengths and diameter of TJ (J), AJ (K) and Des (L) in IBD patients. * P < .05; + P < .01; ++ P < .001

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Experimental details: Figure 2 Confocal micrographs of ARPE-19 cells cultured for 4 months. The ARPE-19 cells were plated on laminin-coated Transwell (r) filter membrane at a density of 3 x 10 5 cells/cm 2 and grown for 4 months as outlined in the Methods section. Rabbit anti- zonula occludens-1 (ZO-1), mouse anti-premelanosome protein (PMEL), mouse anti-claudin-2 primary antibodies, and Alexa Fluor secondary antibodies were used to immunostain the ARPE-19 cells at 4 months post-confluency; rhodamine-phalloidin was used to stain actin. A : PMEL immunostaining correlates with the presence of melanosome pigmentation. B : ZO-1 immunostaining shows good junctional development and localization at the cell borders containing a mixture of elongated and polygonal cells. C : Actin immunostaining reveals circumferential actin distribution throughout the height of the cells. D : Claudin-2 immunostaining shows tight junction localization uniformly across the monolayer. E : ZO-1 immunostaining shows good junctional development and localization at the cell borders regardless of the secondary antibody used. F : Claudin-2 immunofluorescent labeling shows colocalization with ZO-1 in the tight junction complexes (merged image of D and E ). Images were taken using an Andor Revolution XD spinning disk confocal microscope using the 40X Plan Fluor oil immersion objective. Scale bar = 15 mum.

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Experimental details: Fig 6 TLR3 deficiency leads to altered TJ protein expression in OE cell monolayers during Chlamydia infection. WT, TLR3-deficient, and IFN-beta pre-treated TLR3-deficient OE cells were infected with 10 IFU/ cell C . muridarum for up to 36 hours before cell lysates were harvested at the various time-points shown. Protein expression levels were measured by western blot analyses for: (A) ZO-1, occludin, and claudin-1; (B) JAM1 and claudin-2; and (C) claudin-3. OE-129WT = wild-type and OE-TLR3(-) = TLR3-deficient OE cells. Blots shown are representative data . Right-side plots show densitometry of tight-junction proteins normalized to the beta-actin control for the representative data. The relative band intensity is the ratio number of respective band intensity for each TJ protein (minus background) at the indicated time-point, divided by the band intensity for beta-actin (minus background) at that specific time-point shown in the representative western blot data . Significance was determined by comparing the ratio number of the representative data for WT and IFN-beta pre-treated TLR3-deficient OE cells, to the ratio number of OE-TLR3(-) cells divided by beta-actin for three independent experiments. P-values were calculated using a two-tailed unpaired t -test, error bars represent mean (SD) * = p

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Experimental details: Figure 4 Expression of zonula occludens-1 (ZO-1), occludin (OCLN), and claudin-2 (CLDN2) in HFE 145 cell lines after cultured with Helicobacter pylori 43504 strain (HP ATCC 43504; O4 strain). (A) Western blot analysis of ZO-1, OCLN, and CLDN2. (B, D) There were no significant differences in the expression of ZO-1 and CLDN2 between control and H. pylori infection group. (C) The expression of OCLN was significantly elevated 48 hours after the culture with H. pylori strain 43504. Data are expressed as means +- SE for 3 experiments. * P < 0.05 significantly different from the control.

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Experimental details: Figure 3 BmpR1a DeltaFoxL1+ mice displayed abnormalities in goblet cell vesicles and presented dysregulated mucus maturation and structural gene expression. ( A ) Relative mRNA levels of the colonocyte/secretory lineage specification Hes1 and Atoh1 , along with committed-secretory cell toward the goblet fate or maturation Gfi1 , Spdef and Klf4 . A significant decrease was observed for Klf4 mRNA levels in mutant mice while other mRNAs were not modulated between mutant and control mice ( N = 7-9). ( B ) Ultrastructural analysis of the goblet cells revealed vesicles without mucin diversity, disturbed morphology in BmpR1a DeltaFoxL1+ mice ( N = 4). ( C ) Vesicles pattern quantification revealed a significant increase in light grey vesicles in mutant goblet cells when compared to controls ( N = 3). ( D ) BmpR1a DeltaFoxL1+ mice showed a significant increase in Fut2 , Muc2 , Muc4 and Agr2 mRNA levels in mutant mice. ( E ) Ultrastructural analysis of the glycocalyx and the apical junctional complex (yellow bracket) revealed that the latter was not modified between both groups and showed loss of glycocalyx in BmpR1a DeltaFoxL1+ mice ( N = 4). ( F ) Immunofluorescence against ZO-1, claudin-1 and 2 as well as JAM-A revealed no modulation in the tight junction complex proteins between both groups ( N = 3). ( C ) 2-way ANOVA test. Data are presented as the mean +- SD. ( D ) Mann-Whitney test. N: Nucleus; Mu: Mucin vesicles; G: Glycocalyx; AJC: Apical junctional complex. Fold-change was n

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Experimental details: Fig. 1. Cldn2 and Cldn12 confer independent Ca 2+ permeability to the proximal colon. P Ca measured ex vivo in Ussing's chambers across the proximal colon compared to WT of Cldn2 KO ( A ) ( n = 8 per group and P = 0.016); Cldn12 KO ( B ) ( n = 8 per group and P = 0.012); and Cldn2/12 DKO ( C ) ( n = 8 per group and P = 0.019). Full results of bionic dilution potential experiments on small intestine and colon of DKO mice are in SI Appendix , Fig. S2 and Tables S1-S4 . Means were compared by Student's t test. ( D ) Data from A-C expressed as the percentage change in P Ca relative to WT for each genotype. Data are presented as mean +- SD. One-way ANOVA with Dunnett correction for multiple comparisons to compare mean from DKO mice to Cldn2 KO ( P = 0.017) and Cldn12 KO ( P = 0.010) was performed. The Cldn12 coding exon was replaced with a LacZ cassette. ( E and F ) We used this to localize Cldn12 expression by X-gal staining of colon from Cldn12 WT ( E ) and Cldn12 heterozygous mice ( F ). Staining is present in colonic crypt epithelial cells in Cldn12 heterozygous mouse (cyan). (Scale bars, 100 um [ E ] and 25 um [ F ]). ( G ) X-gal staining of colon for Cldn12 (cyan) and immunohistochemical staining for claudin-2 (brown) from a Cldn12 heterozygous mouse. Cr = crypts, SM = smooth muscle, and L = colonic lumen. (Scale bar, 25 um.) * P < 0.05.

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Experimental details: Regulation of CLDN2 protein expression by miR-195-5p in colonic epithelial cell lines. ( A ) Western blot analysis of CLDN2 protein expression in HT-29, Caco2, and T84 cell lines after miR-195-5p mimic transfection. A significant reduction of CLDN2 expression was detected in all cell lines. Raw data of the independent experiments of Western blot were reported in Appendix A . ( B ) Immunofluorescence staining of CLDN2 in HT-29, Caco2 and T84 cell cultures after miR-195-5p mimic transfection. In accordance with Western blot results, after transfection, the expression of CLDN2 decreased compared with mock-control. Single channel images of DAPI and CLDN2 staining before merge were reported in Appendix B . Data of WB were obtained by dividing the normalized transfected sample values by the normalized control sample values. beta-Tubulin was used as housekeeping protein to normalize the data. Data are representative of four independent experiments. The histograms correspond to mean +- SEM. Scale bar = 50 um ** p < 0.01; *** p < 0.001.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 3 Localization of claudin-2 in equine tissues via immunohistochemistry. Scale bar = 100 um.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Analysis of Cldn2 expression and localization in MDCK II cells following quercetin treatment. ( A ) Western blot analysis of Cldn2 expression in cell lysates from control and 400 uM quercetin-treated MDCK II cells. Actin was used as a loading control. ( B ) Densitometry measurements of Cldn2 and actin intensity on western blot were normalized to control at 1 h. Normalized Cldn2 expression relative to normalized actin is plotted (P int = 0.052; P time = 0.096; P treat = 0.0021). ( C ) Immunofluorescence of control and 400 uM of quercetin-treated MDCK II cells at 1, 6, 24, and 48 h. Cldn2 is shown in green and ZO1 is shown in red. ( D ) Localization of claudin at the tight junction was assessed by determining the amount of ZO1 that co-localized with claudin expression (P int = 0.15; P time = 0.46; P treat = 0.0055). For all graphs, each point corresponds to an independent experiment; mean and SEM are shown. C = Control and Q = Quercetin. Scale bar, 20 um.