32-9400

antibody from Invitrogen Antibodies

Targeting: CLDN4 CPE-R, CPETR, CPETR1, hCPE-R, WBSCR8

Provider product page for 32-9400

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Antibody data

Antibody Data
Antigen structure
References [220]
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Validations
Western blot [3]
Other assay [32]

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Product number: 32-9400 - Provider product page
Provider: Invitrogen Antibodies
Product name: Claudin 4 Monoclonal Antibody (3E2C1)
Antibody type: Monoclonal
Antigen: Synthetic peptide
Description: Reactivity has been confirmed with human, rat ileum homogenates, and canine MDCK cell lysates by western blotting and immunofluorescence. Reactivity has also been confirmed with formalin-fixed, paraffin-embedded (FFPE) human normal colon and colon cancer tissues by immunohistochemistry.
Antibody clone number: 3E2C1
Concentration: 0.5 mg/mL

CLDN4 protein structure - 32-9400 shown in red.

Supportive validation

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Experimental details: Western blot analysis of Claudin-4 was performed by loading 20 µg of Mouse Small Intestine (lane1) and PC-3 (lane2) lysates using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (LC5800), and iBlot® Dry Blotting System (IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk at 4°C overnight. Claudin-4 was detected at ~27 kDa using Claudin-4 Mouse Monoclonal Antibody (Product # 32-9400) at 1-3 µg/mL in 2.5 % skim milk for 3 hours at room temperature on a rocking platform. Goat Anti-Mouse IgG - HRP Secondary Antibody (Product # 62-6520) at 1:4000 dilution was used and chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

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Experimental details: Knockdown of Claudin 4 was achieved by transfecting MCF7 with CLDN4 specific siRNAs (Silencer® select Product # s223316, s3441). Western blot analysis (Fig. a) was performed using membrane enriched extracts from CLDN4 knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blot was probed with Claudin 4 Monoclonal Antibody (3E2C1) (Product # 32-9400, 1.5 µg/ml) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to CLDN4..

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Experimental details: Western blot was performed using Anti- Claudin 4 Monoclonal Antibody (3E2C1) (Product # 32-9400) and a 18 kDa band corresponding to CLDN4 was observed across the positive cells lines tested (MCF7 and HT-29) and not in the negative models (A549 and HeLa) as reported. Membrane enriched extracts (30 µg lysate) of MCF7 (Lane 1), HT-29 (Lane 3), A549 (Lane 2) and HeLa (Lane 4) were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX). 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) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). [doi: 10.3892/ijo.2013.1881].

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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 1 Comparison of expression levels of claudins- 1 (1A), 2 (1B), 4 (1C) and 7(1D) in Misoprostol treated (M+) (samples 1+ to 7+) versus untreated (M-) (samples 1- to 5-) cervix . Representative immunoblots in the top panels whilst box plots, lower panel, represent mean (SE) relative optical densities (ROD) for all studied tissues: M+ (n = 10) vs. M- (n = 5). Block arrows showing Gbetaeta as internal loading control at 35 kDa. C-positive control in the first lane. C-1 = claudin-1; C-2 = claudin-2; C-4 = claudin-4; C-7 = claudin-7. ROD- relative optical density in Arbitrary Units (AU).

Supportive validation

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Experimental details: Fig 4 Effects of claudin-2 knockout on the localization of other claudins. (A) Immunofluorescence analysis of claudins in co-culture of control MDCK II cells and claudin-2 knockout clone 1 (KO 1). Claudin-1, -3, -4, and -7 showed clearer and stronger signals at TJs in claudin-2 knockout cells than in control cells. Scale bar = 10 mum. (B) Quantification analysis of signal intensity of claudins at TJs in control MDCK II cells and claudin-2 knockout clones. The signal intensity of claudins at TJs in control cells and claudin-2 knockout clones was measured as described in Materials and Methods , and the relative signal intensity of each claudin was calculated as the ratio of the signal intensity in control cells (CTL) and claudin-2 knockout clones (KO 1 and 2) to the signal intensity in control cells. N = 4 for each experiment. * p < 0.05, ** p < 0.01 compared with control.

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Experimental details: Fig 5 Localization of claudins in z-axis plane in control and claudin-2 knockout cells. Immunofluorescence analysis of claudins and occludin in co-culture of control MDCK II cells and claudin-2 knockout clone 1 (KO 1) in z-axis plane. The signals of claudin-1, -3, -4 and -7 were stronger at TJs in claudin-2 knockout cells compared with control cells, and lateral localization of these claudins was similar between control and claudin-2 knockout cells. Scale bar = 5 mum.

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Experimental details: Fig 8 Effects of claudin-2 re-expression on the localization of other claudins in claudin-2 knockout cells. (A) Immunofluorescence analysis of claudins and FLAG in co-culture of claudin-2 knockout cells and F2 or F4 clones. Claudin-1, -3, -4, and -7 staining at TJs in F2 and F4 clones was weaker than those in claudin-2 knockout cells. Scale bar = 10 mum. (B) Quantification analysis of the signal intensity of claudins at TJs in F2 and F4 clones. The signal intensity of claudin-1, -3, -4, and -7 at TJs in F2 and F4 clones was compared with that in claudin-2 knockout cells. N = 4 for each experiment. * p < 0.05, ** p < 0.01 compared with claudin-2 knockout cells.

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Experimental details: Fig 11 Localization of claudins at TJs between control and their respective knockout cells. (A-E) Immunofluorescence analysis of claudins and ZO-1 or ZO-3 in MDCK II cells transfected with the TALEN constructs for claudin-1, -2, -3, -4, or-7 gene knockouts. After transfection, cells were subcultured on filter inserts for 4 days before the analysis of claudin-2 and -3 (B and C), and for 2 days before the analysis of claudin-1, -4, and -7 (A, D, and E). (F) Immunofluorescence analysis of claudin-2 and ZO-1. TALEN constructs for the claudin-2 gene knockout were transfected into MDCK II cells, which were subcultured on filter inserts for 2 days before analysis. (G) Immunofluorescence analysis of claudin-2 and ZO-1 in a co-culture of wild-type and claudin-2 expressing MDCK I cells. Cells were cultured on filter inserts for 4 days before analysis. Scale bar = 10 mum.

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Experimental details: Figure 4 Western blot and immunofluorescence analysis of parental bipotent cells and MPCs using various markers from the signature of claudin-low breast cancer subtype. ( A ) Western blot analysis of cell lysates from exponentially proliferating bipotent cells and MPCs were analyzed for expression of indicated markers using specific antibodies. Breast cancer cell lines MDA-MB-231, MCF-7, and Hs578T were used as controls. ( B ) Immunofluorescence staining using CD24 antibody (red), nuclei (blue) represent DAPI staining.

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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 2 Cldn6 induces the formation of cell-cell junctions and apicobasal cell polarity in F9 stem cells. (A and D) X-Y and Z projections of F9:Cldn6 cells stained for Cldn6 together with the tight-junction markers (Cldn7, Ocln, ZO-1, and ZO-1alpha+ variant), the basolateral maker (E-Cad), and the apical markers (ezrin, radixin, and EBP50). The arrowhead indicates the recruitment of Ocln to a part of Cldn6-positive premmature cell-cell junctions. The asterisk shows the absence of EBP50 on the apical surfaces of undifferentiated cells possessing Cldn6-positive premature cell-cell junctions. Scale bars, 20 um. (B) Confocal images of undifferentiated areas of F9:Cldn6 cells stained for Cldn6 together with Ocln. The arrowheads indicate the partial recruitment of Ocln to Cldn6-positive cell borders of undifferentiated cells. Scale bar, 20 um. (C) Confocal images of F9:Cldn6 cells stained for Cldn6 together with Cldn4 and DAPI. Scale bar, 20 um.

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Experimental details: Figure 4. Expression and localization of tight junction (TJ) proteins in cell lines expressing typical hepatocyte polarity. Fully polarized cultures were examined. ( A ) western blot. Each lane was loaded with a cell lysate aliquot containing 25 mug total protein. ( B ) RT-PCR. For western blot and RT-PCR, experiments were performed three times with lysates or RNA preparations from two to three independent cultures; one representative experiment is shown. ( C ) Summary of the localization of ten TJ proteins. Experiments were performed at least three times on three independent cultures. ( D ) Images of the localization of some TJ proteins; for each case, the corresponding phase-contrast image is given on the left. Some bile canaliculi (BC) are indicated by arrows (short arrows for BC formed between two cells, long arrows for extended BC). Bar, 10 mum

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Experimental details: Figure 4 Quantitative cell targeting using QD-FcR-Ab conjugates. (A) phaseand fluorescence images of Panc-1 cells incubated with different concentrationsof QD-FcR-aCLDN4 conjugates. (B) The fluorescence of known concentrationsof QD suspensions is used for calibration (see Materialsand Methods for details). (C) Calibration curve showing averagefluorescence intensity per unit area versus QD concentration per unitarea. The calibration curve is used to convert the fluorescence intensityper unit area on the cell surface to an expression level per unitarea. (D) Average fluorescence intensity per unit area for CLDN4 expressionon Panc-1 cells versus QD concentration. Error bars represent standarderror. Each point represents the average intensity from at least 200cells.

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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 3 Tight junction proteins occludin and claudin-4 are co-expressed in Xenopus corneal epithelium lateral membranes and are disrupted at night. Double-label confocal immunocytochemistry was performed on whole flat-mounted preparations of Xenopus corneas that were obtained from animals in the late afternoon (DAY; 9 hours after lights on in a 12L:12D cycle) and in the late night (NIGHT; 3 hours before lights on). ( A ) In the late afternoon, occludin (green) was uniformly primarily localized to the lateral membranes of the surface CE. ( B ) The same specimen as in A was labeled for the presence of claudin-4 (red), and was also uniformly primarily localized to the lateral membranes of the surface CE. ( C ) Merged green/red images from A and B demonstrate a high degree of co-localization in the surface cell CE lateral membranes, as indicated by the yellow signal. ( D ) In the late night, occludin (green) was localized to the CE surface cell lateral membranes as during the day, but the pattern of labeling was often interrupted in some clusters of cells (arrows). ( E ) The same specimen as in D was labeled for the presence of claudin-4 (red), and was also localized to the CE surface cell lateral membranes, but the pattern of labeling was also interrupted in the same clusters of cells (arrows). ( F ) Merged green/red images from D and F demonstrate a high degree of co-localization in the surface cell CE lateral membranes, as indicated by the yellow signal, with a similar level

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Experimental details: Figure 5 Claudin-4 expression on Xenopus surface CE is disrupted at night, and is inversely associated with MMP-2 expression. Flat-mounted whole corneas were double-labeled for localization of claudin-4 and MMP-2 immunoreactivity in the late afternoon (DAY; 9 hours after lights on in a 12L:12D cycle) and in the late night (NIGHT; 3 hours before lights on). ( A ) In the late afternoon, claudin-4 (green) immunolabeling of the lateral membranes of the surface CE was generally intact, and some sporadic labeling of MMP-2 (red) was also present on the lateral membranes and/or cytoplasm (arrows) which was sometimes co-localized (yellow) with occludin. ( B ) During the late night, most of the surface CE cells exhibited intact claudin-4 (green) labeling on their lateral membranes, but there were also many clusters of surface CE cells in which the claudin-4 immunoreactivity was disrupted (arrows) and was accompanied by high levels of MMP-2 immunoreactivity. Specimens were stained with the blue nuclear DAPI stain. Scale bar = 20 um.

Supportive validation

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Experimental details: Figure 7 Membrane type 1-MMP is expressed in surface CE cells and associates with lateral membranes at nighttime. Flat-mounted whole corneas were double-labeled for localization of claudin-4 and membrane type 1-MMP (MT1-MMP) immunoreactivity in the late afternoon (DAY; 9 hours after lights on in a 12L:12D cycle) and in the late night (NIGHT; 3 hours before lights on). ( A ) In the late afternoon, claudin-4 (red) immunolabeling of the surface CE cell lateral membranes was generally intact and uniform. MT1-MMP (green) labeling, when present, was observed almost exclusively in cytoplasmic compartments of the surface CE. ( B, C ) Two examples are presented to illustrate that during the late night, a majority of the surface CE cells exhibited intact claudin-4 (green) labeling on their lateral membranes, but there were also many clusters of surface CE cells in which the claudin-4 immunoreactivity was disrupted. Also at nighttime, MT1-MMP labeling was present both in the cytoplasm and intermittently associated with the surface CE cell lateral membranes (arrows). The membrane co-localization of the red claudin-4 and green MT1-MMP resulted in a merged yellow labeling of the lateral membranes in some areas (arrows). Specimens were stained with the blue nuclear DAPI stain. Scale bar = 20 um.

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Experimental details: Figure 3 Protein expression (Western blot) of claudin-1, claudin-3, claudin-4, claudin-5, claudin-7, and claudin-8 after treatment with bacterial strains (Ecf, ETEC, and Ecf + ETEC). (a) Protein expression relative to respective controls [means +- SEM]. Samples were taken after 8 h. No differences were observed between treatment groups. N = 3 independent experiments per bar. (b) Exemplarily, data of one Western blot is shown. 1 = control (8 h), 2 = Ecf (8 h), 3 = ETEC (8 h), 4 = Ecf (8 h) + ETEC (6 h), and 5 = Ecf (10 h) + ETEC (8 h). Sample 4 was included as a control to rule out effects of longer total bacterial incubation (preincubation) and was not included in the statistical analysis shown in (a).

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Experimental details: Figure 12 Effects of ZO-1 knockout on the localization and expression levels of claudins. (A) Effects of ZO-1 knockout on the localization of claudins. Control and ZO-1 knockout cells of clone 1 were co-cultured on filter inserts. Signal intensity of claudins on lines shown in confocal microscopic images ( arrows ) were analyzed. Claudin-2 fluorescent signal at TJs was increased but claudin-1 and -7 signals at TJs were reduced in ZO-1 knockout cells. Scale bar, 10 um. (B) Immunoblots of claudins in control MDCK II cells and ZO-1 knockout clones. Similar expression levels of claudins were observed in control and knockout cells.

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Experimental details: Fig 3 Effects of claudin-2 knockout on the localization of other TJ proteins and cytoskeleton. (A) Immunofluorescence analysis of claudin-1, -3, -4, -7, ZO-1, occludin, F-actin, and myosin heavy chain II-B (MHC-B) in control and claudin-2 knockout cells. Claudin-1 and -7 showed a tendency to be more clearly localized at TJs in claudin-2 knockout cells. Scale bar = 10 mum. (B) Immunoblots of claudin-1, -3, -4, and -7 in control MDCK II cells and claudin-2 knockout clones. Similar expression levels of claudin-1, -3, -4, and -7 were observed in control cells and knockout clones.

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Experimental details: Figure 1. Examples of CD24, CD44 and claudin 4 and 7 immunostaining in mammary invasive carcinomas (scale bar, 100 um). (A) Claudin-negative; (B) CD24-negative; (C) CD44-negative; (D) claudin 4 2+; (E) claudin 7 2+; (F) CD24-positive; (G) claudin 4 3+; (H) claudin 7 3+; (I) CD44-positive.

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Experimental details: Figure 7 rEhCP112a degrades claudin-1 and increases claudin-2 expression in Caco-2 cells. (A) Caco-2 cells incubated with rEhCP112a for different times or with live trophozoites (T) for 10 min, submitted to WB using antibodies against TJ proteins indicated at left and alpha-tubulin as a loading control. (B) Densitometry analysis of bands obtained in (A) for each TJs protein with regards to tubulin. * p

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Experimental details: Figure 8 rEhCP112p binds to claudin-1 and claudin-2. Confluent Caco-2 cell monolayers were incubated for 10 min with rEhCP112p, then, cell lysates were immunoprecipitated (IP) in the presence of protease inhibitors, using alpha-EhCP112 antibody or pre-immune serum (PS). Immunoprecipitates, rEhCP112p and Caco-2 cell lysates (Caco-2) were submitted to WB assays and revealed using the antibodies indicated at left.