51-9000

antibody from Invitrogen Antibodies

Targeting: CLDN1 ILVASC, SEMP1

Provider product page for 51-9000

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Antibody data

Antibody Data
Antigen structure
References [200]
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Product number: 51-9000 - Provider product page
Provider: Invitrogen Antibodies
Product name: Claudin 1 Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Reactivity: Human, Rat, Chicken/Avian
Host: Rabbit
Isotype: IgG
Vial size: 100 µg
Concentration: 0.25 mg/mL
Storage: -20°C

CLDN1 protein structure - 51-9000 shown in red.

Supportive validation

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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.

Supportive validation

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Supportive validation

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Supportive validation

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Supportive validation

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Supportive validation

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Experimental details: Fig. 9 Localization of claudin-1: photomicrographs showing serial sections of human renal cortical tissue stained for ZO-1 ( A ), calbindin ( B ), aquaporin-2 ( C ), claudin-1 ( D ), THP ( E ) and L. tetragonolobus ( F ) [claudin-1-, calbindin- and aquaporin-2-stained coincident tubules that were negative for THP and L. tetragonolobus (arrowheads in B-F); there were some calbindin positive tubules that were negative for claudin-1, THP and aquaporin-2 (asterisks in B-E); claudin-1 basolateral membrane location, corresponding with tubules showing discrete junctional ZO-1 staining (arrowheads in A and D); robust ZO-1 staining was seen in glomerular epithelial cells (A); scale bar 100 um].

Supportive validation

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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

Supportive validation

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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.

Supportive validation

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Main image
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.

Supportive validation

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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.

Supportive validation

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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.

Supportive validation

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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

Supportive validation

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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

Supportive validation

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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

Supportive validation

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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).

Supportive validation

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Experimental details: Fig. 6 Claudin-5 and claudin-4 are expressed in endothelial cells in the developing brain a - d . Cld-5 immunoreactivity in the developing and adult brain. Parenchymal vessels are labeled ( green ) in E19 ( arrows a ) or adult ( arrows b , d ) animals. In c , the anti-Cld-5 polyclonal antibody was used to show the colocalization of the TJ protein ( red ) with the endothelial marker RECA-1 ( green ) in a P2 animal. In all CPs immunodetection revealed a signal associated with the largest vessels penetrating the choroidal stroma ( arrowheads b , d ), but not with the choroidal epithelium, labeled in red by anti-Cld-1 Ab ( d ). Note that all cisternal vessels such as the vein of Galen are also labeled by anti-Cld-5 Ab. DAPI was used for nuclei staining. e Representative Western blot of Cld-4. Brain cortex ( lane 1 20 mug) and MV ( lanes 2 and 3 40 mug) were isolated from adult animals. Lane 4 was loaded with MDCK (10 mug) as positive control. A low, but specific Cld-4 signal was detected in MV ( upper panel lane 3 band at 22 kDa), but not in cortex or in MV when the primary antibody was omitted ( lane 2 ). Actin used as a loading indicator is shown in the lower panel (42 kDa band). Due to the high expression of Cld-4 in MDCK cells, lane 4 was treated separately from the other lanes for HRP detection. f - h Cld-4 immunoreactivity ( red ) in the developing and adult brain. Double immunolabeling with anti-RECA-1Ab ( green ) allowed visualizing the parenchymal vessels that were posit

Supportive validation

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Experimental details: Fig. 1 Knockdown of claudin-7 in HCC827 lung cancer cells. a Representative phase and green fluorescence images of live control and claudin-7 KD cells. Both control and claudin-7 shRNA lentivirus constructs contain a GFP expression sequence. b Top panel is the anti-claudin-7 immunofluorescence staining of control and claudin-7 KD cells. Claudin-7 level was dramatically decreased in claudin-7 KD cells. The bottom panel is the anti-GFP immunofluorescence staining. The cells were fixed with 100 % methanol, which lead to GFP proteins leaking out of the cells so that the green fluorescence shown in the top panel was only the claudin-7 signal. c Western blot shows the diminished level of claudin-7 in the KD cells ( left ). The statistical analysis of claudin-7 expression in control and KD cells from five independent experiments is shown on right. * P < 0.05. d Protein expression levels of E-cadherin, claudin-1, -3, and -4 in control and KD cells. Only claudin-4 expression level was increased in KD cells. GAPDH served as a loading control

Supportive validation

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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.

Supportive validation

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Experimental details: Fig. 2 Phenotype and irreversible de-differentiation of Immortomouse (r) CPECs. a Primary Immortomouse (r) choroid plexus epithelial cells (p0) grown at 37 degC for 7 days showed the same staining patterns for cytokeratin (CK) and claudin-1 (Cldn-1) as pmCPECs isolated from wild-type mice. b When Immortomouse (r) CPECs were grown under permissive conditions (33 degC, 10U IFNgamma) the heterogenous de-differentiation process started in areas with low cell density, whereas CPECs kept their cuboidal shape longer in areas with high cell density. The contrast of the pictures in a and b was enhanced using Adobe Photoshop software. c Vimentin staining, rather than CK staining was observed at 33 degC. Cell death took place at 33 degC upon Ara-C addition to culture. d The irreversible loss of CPEC specific markers Cldn-1 and CK and an increasing proliferation rate of de-differentiated Immortomouse (r) CPECs was observed with increased passage. The first row is passage 2 (p2), the second row is p3, the third row is p4. e The cells failed to form confluent monolayers or display the correct expression pattern of epithelial markers upon shift to non-permissive temperature and IFNgamma withdrawal. Immortomouse (r) CPECs in e were stained after 7 days of non-permissive growth; the passage numbers (p) were E-cad/DAPI: p8, ZO1/beta-Cat/DAPI: p5, Cldn11/N-Cad/DAPI: p6, Cldn-1/CK/DAPI: p4. Scale bar in all immunofluorescent and phase contrast pictures = 100 mum. p0 primary culture, E-Cad E-cadh

Supportive validation

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Experimental details: Fig. 4 Phenotype of ECPC4 cells and primary mouse choroid plexus epithelial cells (pmCPECs). Immunofluorescence staining for CPEC specific proteins is shown in ECPC4 cells ( a ) and pmCPECs ( b ). a There is weak staining for the adhesion junction (AJ) protein E-Cadherin (E-Cad) and its cytoskeleton linker beta-catenin (beta-Cat) of ECPC4 cells and their localization is not specifically at the plasma membrane. Staining for tight junctional (TJ) claudins-1 and -11 was absent or showed a weak cytosolic pattern, respectively. The scaffolding protein ZO1 staining was disrupted. Additionally, the cell line failed to stain for the early epithelial marker cytokeratin but rather was positive for the mesenchymal intermediate filament protein vimentin. ECPC4 cells from passage 41 were stained on d4 in culture. b In contrast, the staining of pmCPECs stained on d7 in culture, revealed a proper distribution of all epithelial markers. pmCPECs. All staining was performed at least 3 times. Scale bars 50 mum. E-Cad E-cadherin, Cldn-1 Claudin 1, Cldn-11 claudin 11, CK cytokeratin, beta-Cat beta-catenin, ZO-1 zona occludens protein 1

Supportive validation

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Experimental details: Figure 3 MMP13 contributes to tight junction destabilization Source data is available for this figure in the Supporting Information. A-C. Western blot quantification of the protein expression levels of ZO-1 (A), occludin (B) and claudin (C) in mucosal scrapings of ilea from MMP13 +/+ (black) and MMP13 -/- (grey) mice 0 and 8 h after LPS injection ( n = 3-4). D,E. Representative ZO-1 immunogold-labeled TEM images of MMP13 +/+ (D) and MMP13 -/- (E) mice 8 h after LPS injection. F. Schematic overview of the separation of the lipid raft and non-lipid-raft fractions by ultracentrifugation. G. Caveolin-1 western blot analysis of fractions obtained after ultracentrifugation of mucosal scrapings from MMP13 +/+ and MMP13 -/- mice 0 and 8 h after LPS challenge.

Supportive validation

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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

Supportive validation

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Experimental details: Figure 3 Immunomodulation that reduces systemic Treg levels activates the CP for mo-MPhi trafficking and mitigates AD pathology. ( a ) mRNA expression levels of ifn-gamma , measured by RT-qPCR, in CPs isolated from 6- and 12-month-old APP/PS1 AD-Tg mice, compared with age-matched WT controls ( n =5-8 per group; Student's t -test). ( b , c ) 5XFAD AD-Tg mice were treated with either weekly GA or vehicle (PBS), and were examined at the end of the 1st week of the administration regimen (after a total of two GA injections). Flow cytometry analysis of CD4 + Foxp3 + splenocyte frequencies ( b ) and CP-resident IFN-gamma-expressing immune cells (intracellularly stained and pre-gated on CD45) ( c ), in treated 6-month-old AD-Tg mice, compared with age-matched WT controls ( n =4-6 per group; one-way ANOVA followed by Newman-Keuls post hoc analysis). ( d ) mRNA expression levels for the genes icam1 , cxcl10 and ccl2 , measured by RT-qPCR, in CPs of 4-month-old AD-Tg mice, treated with either weekly GA or vehicle, and examined either at the end of the 1st or 4th week of the weekly GA regimen ( n =6-8 per group; one-way ANOVA followed by Newman-Keuls post hoc analysis). ( e ) Representative microscopic images of 6-month-old AD-Tg mice following weekly GA, stained for ICAM-1 (in red) and Claudin-1 (in green; epithelial tight junctions), showing elevated levels of ICAM-1 immunoreactivity, as compared with vehicle-treated AD-Tg (scale bar, 50 mum). ( f ) Representative images of brain