37-4900

antibody from Invitrogen Antibodies

Targeting: CLDN1 ILVASC, SEMP1

Provider product page for 37-4900

Western blot

Immunocytochemistry

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [89]
Comments [0]
Validations
Western blot [2]
Immunocytochemistry [1]
Immunohistochemistry [2]
Other assay [55]

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Product number: 37-4900 - Provider product page
Provider: Invitrogen Antibodies
Product name: Claudin 1 Monoclonal Antibody (2H10D10)
Antibody type: Monoclonal
Antigen: Synthetic peptide
Description: Reactivity has been confirmed with human Caco-2 and canine MDCK cell lysates, mouse kidney and intestinal lysates, frozen mouse intestine (jejunum) tissue, and rat kidney homogenates.
Antibody clone number: 2H10D10
Concentration: 0.5 mg/mL

CLDN1 protein structure - 37-4900 shown in red.

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Experimental details: Western blot analysis of Claudin-1 was performed by loading 20 µg of MDCK (lane1), Caco-2 (lane2), A431 (lane3), A549 (lane4) and HeLa (lane5) cell 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-1 was detected at ~18 kDa using Claudin-1 Mouse Monoclonal Antibody (Product # 37-4900) 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: Western blot was performed using Anti-Claudin1 Polyclonal Antibody (Product # 37-4900) and a 20 kDa band corresponding to CLDN1 was observed across cell lines tested and increased upon EGF & LiCl, IL-beta, and TNFα & TGF-β treatment. Membrane enriched extracts (30 µg lysate) of Caco-2 (Lane 1), Caco-2 treated with EGF (100 ng/mL) and LiCl (50 mM) simultaneously for 24 hr (Lane 2), Caco-2 treated with IL-beta (10 ng/mL for 48 hr) (Lane 3) and 25 µg lysate of A549 (Lane 4) and A549 treated with TGF-βbeta; (10 ng/mL) & TNFα (20 ng/mL) simultaneously for 72 hr 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 µg/mL) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4,000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

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Experimental details: Figure 4 Evidence for morphologic and phenotypic changes consistent with in vitro EMT. A. Phase contrast or immunofluorescence images of DKAT cells cultured in either MEGM (upper) or SCGM for 14 days (lower). Phase contrast images were acquired with a 20 x objective, scale bar = 40 um. IF images were acquired with a 40 x objective, scale bar = 20 um. Cells were stained simultaneously for vimentin (green) and E-cadherin (red), or CK18 (green) and CK5 (red). B. E-cadherin immunofluorescence images of DKAT cells cultured in MEGM or SCGM for 2 hours. C. Total cell lysates from DKAT cells cultured in MEGM or SCGM for 1 h, 24 h, or 14 days were analyzed by western blot for vimentin, E-cadherin and actin. D. Scratch wound healing assay comparing DKAT cells grown in MEGM or SCGM (14 days) and MDA-MB-231 cells. Images (upper panel) were taken at the time of the scratch (T = 0) and 12 hours later with a 5 x objective. Scale bar = 200 um. Results (lower panel) are expressed as the percentage of the scratch area closed at 12 hrs as measured by ImageJ software, and are an average of 12 scratches per sample. E. Total cell lysates from a clonal DKAT cell line cultured in MEGM or SCGM (14 days) were analyzed by western blot for vimentin, E-cadherin and actin. F. Total cell lysate from passage matched cultures of DKAT cells grown in MEGM, SCGM, or SCGM for 10 passages and then returned to MEGM for 5 passages were analyzed by western blot for epithelial and mesenchymal markers. G. Immunofluore

Supportive validation

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Experimental details: Fig. 1 Protein levels of claudin-1, -2, -4 and -5 in equine tissues. Levels were measured by western blot (A) and were normalized against GAPDH protein (B-E; claudin-1, -2, -4 and -5, respectively). The signal intensity of each band was measured using the NIH Image J software (USA). Data are presented as the means +- standard error of the mean of four equine samples. Liv, Liver; Kid, Kidney; Lu, Lung; Duo, Duodenum; Hrt, Heart; Tes, Testis; Ov, Ovary; Ut, Uterus. * p < 0.01 vs . liver; + p < 0.01 vs . kidney; ++ p < 0.01 vs . lung; SS p < 0.01 vs . duodenum; p < 0.01 vs . heart; P p < 0.01 vs . ovary; ** p < 0.05 vs . uterus.

Supportive validation

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Experimental details: Figure 4 HCV pseudo-particles (HCVpp) with envelope proteins of SB strain infected B lymphocytes, but not hepatic cells. ( a ) Claudin1 (CLDN1) or Occuludin (OCLN) expression confers weak susceptibility of Raji cells to JFH1 infection (Upper panel). Immunoblot analysis demonstrated that Claudin-1 and Occludin proteins were expressed by transfection of plasmids. ( b ) The respective expression vectors of CLDN1 and OCLN were transfected into both Raji and Huh7.5.1 cells. Luciferase-encoding pseudo-particles bearing the indicated envelope proteins were used to infect recipient Raji cells transfected with control vector (white), CLDN1-expressing vector (grey), or OCLN-expressing vector, or control Huh7.5.1 cells (black). For HCVpp, the respective E1E2 genes are indicated in parentheses. Luciferase assay was performed at 3 days after transduction (* P

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Experimental details: Figure 5 Zeb1 regulates DKAT EMT/MET. A. Total cell lysate was prepared from DKAT-MEGM or DKAT-SCGM cells and subjected to western blot analysis for markers of EMT. B. Total cell lysate was prepared from DKAT-MEGM cells stably transfected with a control vector (V) or vector containing Zeb1 transcript (Zeb1) and subjected to western blot analysis for markers of EMT. C. Scratch wound healing assay comparing DKAT cells grown in MEGM or SCGM as indicated, stably expressing either a non-targeting shRNA sequence or one of two Zeb1-targeting sequences. Results are expressed as the percentage of the scratch area closed at 16 hrs as measured by ImageJ software, and are an average of 12 scratches per sample.

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Experimental details: Figure 2 Bs 29784 increases the expression of tight junction's proteins. Caco-2 cells were left untreated (white columns) or were treated apically for 16 h with DON at 100 muM (gray columns) or Bs 29784 at an initial bacterial density of 10 7 bacteria/ml (black columns). ZO-1, occludin, and claudin-1 proteins were visualized by Western-blot (A) . Band densities were measured using Image J software and subjected to statistical analysis (B) . * and ** Significant differences between the groups, p < 0.05 and p < 0.01, respectively.

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Experimental details: Figure 5 Effect of TF3'G and cytochalasin D on the transport of gamma-EV across Caco-2 monolayer cells. ( A ) The transport of 1 mM gamma-EV, assessed over a period of 2 h, after the pretreatment of TF3'G (20 muM, 24 h). ( B ) Immunofluorescence staining of the tight-junction proteins ZO-1 and claudin-1 of the TF3'G-treated Caco-2 monolayer cells on the membrane, to ensure the maintenance of cell integrity after 2 h of transport. ( C ) The transport of 1 mM gamma-EV, assessed over a period of 2 h, after the pretreatment of cytochalasin D (0.5 mug/mL, 30 min). ( D ) Immunofluorescence staining of the tight-junction proteins ZO-1 and claudin-1 of the cytochalasin-treated Caco-2 monolayer cells on the membrane, to ensure the maintenance of cell integrity after 2 h of transport. Control immunofluorescence images are same as those mentioned in Figure 4 . Data are presented as the mean +- SD from at least n = 3 (TF3'G and cytochalasin D) and n = 4 (control) independent experiments; *, and *** indicate p < 0.05 and p < 0.001, respectively.

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Experimental details: Figure 6 Effect of Gly-Sar on the transport of gamma-EV across Caco-2 monolayer cells. ( A ) The transport of 1 mM gamma-EV, assessed over a period of 2 h, after the pretreatment of Gly-Sar (25 mM, 30 min). ( B ) Immunofluorescence staining of the tight-junction proteins ZO-1 and claudin-1 of the Gly-Sar-treated Caco-2 monolayer cells on the membrane, to ensure the maintenance of cell integrity after 2 h of transport. ( C ) The transport of 1 mM gamma-EV, assessed over a period of 2 h, after the pre-treatment of both TF3'G and Gly-Sar simultaneously. Control immunofluorescence images are same as those mentioned in Figure 4 . Data presented as the mean +- SD from at least n = 3 (Gly-Sar) and n = 4 (control and TF3'G/Gly-Sar) independent experiments; * and ** indicate p < 0.05 and p < 0.01, respectively.

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Experimental details: Fig. 1 Reconstitution of the human substantia nigra Substantia Nigra Brain-Chip. a Schematic depiction of the Substantia Nigra Brain-Chip of a two-channel microengineered chip with iPSC-derived brain endothelial cells cultured on all surfaces of the lower vascular channel, and iPSC-derived dopaminergic neurons, primary human brain astrocytes, microglia, and pericytes on the upper surface of the central horizontal membrane in the upper brain channel. b 3D reconstruction of a confocal z-stack showing the organization of all five cell types in the Substantia Nigra (SN) Brain-Chip. c Representative image of iPSC-derived dopaminergic neurons that are stained with DAPI (blue), microtubule-associated protein 2 (green, MAP2), tyrosine hydroxylase (red, TH), and a merged image on day 8. Scale bars: 100 mum. d Immunofluorescence micrographs of the human brain endothelium cultured on the vascular channel of Brain-Chip for 7 days post-seeding (D8) labeled with Claudin-1 (red), Claudin-5 (cyan), Occludin (yellow), and CD31 (white). Scale bars: 100 mum. e Immunofluorescence micrographs demonstrate high levels of expression of zonula occludens-1 (red, ZO-1) across the entire endothelial monolayer. Scale bars: 100 mum. f Quantitative barrier function analysis via apparent permeability to 3 kDa fluorescent dextran, and 0.5 kDa lucifer yellow (LucY) crossing through the vascular to the neuronal channel on day 5 and 8. Statistical analysis is two-way ANOVA with Tukey's multiple comparisons test

Supportive validation

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Experimental details: Fig. 3 The in ovo injection of methionine enhances the intestinal barrier function of chicks. (A) Transepithelial cell resistance (TEER) of the jejuna of chicks injected with phosphate-buffered saline (PBS) or L-methionine (L-Met). (B to C) The expression of ZO-1 and claudin-1 in the jejunum of chicks injected with PBS or L-Met. (D) Representative images of immunofluorescence (IF) staining with a ZO-1 antibody in the chick jejunum are shown (200x magnification; scale bar, 100 mum). (E) Statistical analysis of the fluorescence signal intensity based on the images shown in (D). (F) Representative images of IF staining with a claudin 1 antibody in the chick jejunum are shown (200x magnification; scale bar, 100 mum). (G) Statistical analysis of the fluorescence signal intensity based on the images shown in (F). Bars without a common letter indicate significant differences ( P < 0.05); the values are means +- SEM. n = 3. CON = control. Fig. 3

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Experimental details: Figure 4. In adult SMGs and PGs, FGF10 pos cells are restricted to the ED, SD, and GCT ducts and express ionocyte markers (A) In adult glands, FGF10 pos cells (TOM, red) express E-cadherin (ECAD, green), KRT7 (green), ASCL3 (green), FOXI1 (green), NKCC1 (green), and CFTR (green), which are expressed at the apical membrane of FGF10 pos cells. Nuclei labeled with DAPI (blue). (B) In adult SMG FGF10 pos cells (TOM, red) were found in the GCTs (red arrows) and SDs (yellow arrows) but not in the IDs (white arrows). (C) FGF10 pos cells were labeled at P29/30 (TOM, red) and stained with different antibodies. Acini were labeled with claudin (orange); MECs were labeled with alphaSMA (green); the basement membrane was labeled with heparan sulfate (HS) (gray); ducts were labeled with pannexin-1 (PANX1); stromal cells were labeled with VIM (green); nuclei labeled with DAPI (blue). (D and E) FGF10 pos cells (TOM, red) were found within ducts of SMGs (D) and PGs (E), while not in SLGs (D). FGF10 pos cells were detected only within the mesenchyme. (F) IMARIS bounding box software was used to analyze shape and size of FGF10 pos cells. (G) Summary of immunostaining used to label different cell types and gland compartments. Also see Figures S4 and S5 , Data S6 .

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Experimental details: Figure 2 KN-93 induces up-regulation of barrier function ( A ) Claudin-1 mAb (Clone 2H10D10) recognized claudin-1, which contributed to the formation of ectopic TJs induced by the treatment with KN-93 (arrows). Scale bar, 5 mum. ( B ) Quantification of the time-dependent effect of KN-93 on the enlargement of TJs. EpH4 cells treated with KN-93 for the indicated times were stained with DAPI and the anti-claudin-1 mAb (2H10D10). The number of cells with extended TJs was determined as a percentage of the total number of cells (based on DAPI staining) in the same field. Each experiment (n = 3) included 100 cells. Data are represented as the mean +- s.d. ( C ) EpH4 cells were treated with DMSO (control) or 10 muM KN-93 for 5 hours and stained with anti-claudin-3 pAb (green) and anti-ZO-1 mAb (red). Scale bar, 20 mum. ( D ) Immunoblotting of whole-cell lysates from EpH4 cells treated with DMSO (control) or 10 muM KN-93 with the indicated antibodies. ( E ) CSG1 cells were cultured in Transwell chambers and treated with DMSO (control) or 10 muM KN-93 for 5 hours and then analyzed for their TER. Analysis of TER showed about a 1.8-fold increase after the treatment with KN-93. Data are mean +- s.d.; *P < 0.05 (Student's t-test).

Supportive validation

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Experimental details: Figure 5 TJs enlarge as an adaptation response to hyperosmotic stress ( A ) CSG1 cells were treated with hyperosmotic medium for 2 hours and doubly stained with anti-claudin-1 pAb (green) and anti-ZO-1 mAb (red) (upper panels), anti-claudin-1 pAb (green) and alpha18 antibody (red) (middle panels) and/or anti-Par-3 pAb (green) and anti-claudin-1 mAb (Clone 2H10D10) (red) (lower panels). Scale bar, 20 mum. ( B ) Z-sections of CSG1 cells treated with hyperosmotic medium. ( C ) Immunoblotting of whole-cell lysates of CSG1 cells treated with normal osmotic medium or 600 mOsm/L medium for 2 hours with the indicated antibodies. ( D ) CSG1 cells stably expressing myc-ubiquitin were cultured in normal medium or hyperosmotic medium for 2 hours. The cell lysates were subjected to immunoprecipitation with anti-myc antibody, followed by immunoblotting with anti-claudin-1 antibody. ( E ) Over-expression of dominant negative AMPK2alpha suppressed the formation of extra TJs, even under hyperosmotic conditions. CSG1 cells were transfected with mCherry only as a control or mCherry-AMPKalpha2 K45M. Then, cells were cultured in hyperosmotic medium for 2 hours. Then, cells were fixed and stained for anti-claudin-1 pAb (green). The percentages were calculated as (the number of cells with normal TJs under hyperosmotic stress)/(the number of cells transfected with mCherry only or mCherry-AMPKalpha2 K45M). In each experiment, the total cell number was 100 (n = 3). Data are mean +- s.d.; *P < 0.05 (St

Supportive validation

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Experimental details: Fig. 2 Localization of claudin-1 in equine tissues via immunohistochemistry. Scale bar = 100 um.

Supportive validation

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Experimental details: Fig. 1 Co-culture with mesenchymal stem cells (MSCs) attenuates TGF-beta1-induced epithelial-mesenchymal transition (EMT) in corneal epithelial cells (CECs). (a) Phase contrast images of CECs with or without TGF-beta1 treatment. Scale bar, 100 mum. (b) Schematic of experimental method. (c) Gene expression analysis of EMT-related markers in CECs with or without co-culture with AdMSC (10,000 or 20,000 cells/insert). Data are expressed as the means +- SEM; n = 4 cell samples; *p < 0.05, **p < 0.01, and ***p < 0.001. (d) Immunostaining for VIM (red) and CLDN1 (green) in CECs. Nuclei, blue; scale bars, 100 mum. Fig. 1

Supportive validation

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Experimental details: Fig. 3 Conditioned medium from Adipose-derived mesenchymal stem cells (AdMSC-CM) attenuates TGF-beta1-induced epithelial-mesenchymal transition (EMT) in corneal epithelial cell (CEC) sheets. (a) Schematic of experimental method. (b) Immunostaining for VIM (red) and CLDN1 (green) in CEC sheets at the apical and basal region. Nuclei, blue. Scale bar, 50 mum. (c) Immunostaining for VIM (red) and CLDN1 (green) in whole-mounts of CEC sheets constructed as a three-dimensional image. Nuclei, blue. Scale bars, 20 mum. Fig. 3

Supportive validation

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Experimental details: Figure 1 Constitutive expression of EGFP-EspF and EGFP-Map in MDCK cells disrupts the tight junction barrier. ( A ) Untransfected cells or cells constitutively expressing EGFP vector alone, EGFP-Tir, EGFP-EspF and EGFP-Map were cultured on polycarbonate Transwell filters and TER was measured daily to monitor the integrity of tight junctions. The maximum values of TER for each cell line are indicated. The paracellular flux of 4 kDa dextran ( B ) and 70 kDa dextran ( C ) was measured and plotted as fold change with respect to untransfected MDCK cells (normalized to 1). Error bars represent means +- s.e.m from three independent experiments; *indicates p-value < 0.01 and **indicates p-value < 0.001. ( D ) The cellular localization of TJ proteins was examined using the indicated primary antibodies and Cy3-conjugated secondary antibodies. Nucleus was labeled with DAPI (4',6-Diamidino-2-phenylindole dihydrochloride). Constitutive expression of EGFP-EspF and EGFP-Map caused the reorganization of claudin-1, -4, occludin and ZO-1. Scale: 10 um; UT: Untransfected.

Supportive validation

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Experimental details: 6 Upregulation of the Notch signaling components in podocytes and/or renal progenitors of SCID mice with adriamycin nephropathy. ( A ): Time course assessment of albumin/creatinine ratio as measured in mice with adriamycin nephropathy (black square) in comparison with PBS-treated mice (red circle). Data are expressed as mean +- SEM as obtained in three independent experiments ( n = 12 mice at each time point for each group of treatment); *, p < .05. ( B ): Double label immunofluorescence for Notch1 (green) and Claudin-1 (Cla1, red); Notch1 (green) and WT1 (red); Notch3 (green) and cytokeratin (CK, red); Notch3 (green) and WT1 (red) in SCID mice affected by adriamycin nephropathy at different time points. Topro-3 counterstains nuclei (blue). One representative of three independent experiments performed on a total of 12 mice is shown. Scale bar = 20 mum. Abbreviations: PBS, phosphate buffered saline; SCID, severe combined immunodeficient.

Supportive validation

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Experimental details: Figure 2 Analysis of Cldn1 expression and localization in MDCK II cells following quercetin treatment. ( A ) Western blot analysis of Cldn1 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 Cldn1 and actin intensity on western blot were normalized to expression at 1h. Normalized Cldn1 expression relative to normalized actin expression is plotted (P int = 0.11; P time = 0.022; P treat = 0.16). ( C ) Immunofluorescence of control and 400 uM of quercetin-treated MDCK II cells at 1, 6, 24, and 48 h. Cldn1 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 was co-localized with claudin expression (P int = 0.24; P time = 0.35; P treat = 0.055). For all graphs, each point corresponds to an independent experiment; mean and SEM are shown. C = Control and Q = Quercetin. Scale bar, 20 um.

Supportive validation

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Experimental details: Figure 2 Expression of TJ proteins in the intestine of rats from different experimental groups: ES--rats were orally gavaged with Saccharomyces cerevisiae fermentate and exposed to heat stress; EC--rats were orally gavaged with Saccharomyces cerevisiae fermentate and kept at room temperature; PS--rats were orally gavaged with PBS and exposed to heat stress; PC--rats were orally gavaged with PBS and kept at room temperature; * P < 0*05.

Supportive validation

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Experimental details: Figure 3 Immunofluorescence staining of TJ proteins in the intestine of rats from different experimental groups. (a) Images of occludin immunostaining, Bar 50 um; (b) Analysis of occludin expression with immunostaining; (c) Images of ZO-1 immunostaining, bar 50 um; (d) Analysis of ZO-1 expression with immunostaining; (e) Images of claudin immunostaining, bar 10 um; (f) Analysis of claudin expression with immunostaining; (g) Images of JAM-A immunostaining, bar 10 um; (h) Analysis of JAM-A expression with immunostaining. A.U., arbitrary units. Rats were pretreated by oral gavage with B. subtilis BSB3 (BSB3) or with PBS and were either exposed forced running or remained sedentary ( n = 6 rats per condition). BEx, pretreated with BSB3, undergoing forced running; BCont, pretreated with BSB3 remaining sedentary; PEx, pretreated with PBS, undergoing forced running; PCont, pretreated with PBS remaining sedentary. Data with different superscript letters signify groups are significantly different ( P < 0*05). [Colour figure can be viewed at https://www.wileyonlinelibrary.com ]