38-9100

antibody from Invitrogen Antibodies

Targeting: TJP2 DFNA51, X104, ZO-2, ZO2

Provider product page for 38-9100

Western blot

Immunocytochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [20]
Comments [0]
Validations
Western blot [4]
Immunocytochemistry [3]
Other assay [17]

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Product number: 38-9100 - Provider product page
Provider: Invitrogen Antibodies
Product name: ZO-2 Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Reactivity: Human, Mouse, Rat, Canine
Host: Rabbit
Isotype: IgG
Vial size: 100 µg
Concentration: 0.25 mg/mL
Storage: -20°C

TJP2 protein structure - 38-9100 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of (A) dog MDCK, (B) human Caco-2, (C) rat KNRK, and (D) mouse TCMK-1 cell lysates using Zymed (R) Rb anti-ZO-2 (C-term) (Product # 38-9100).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of A431 (Lane 1) and U-2 OS (Lane 2). The blots were probed with Anti- ZO-2 Rabbit Polyclonal Antibody (Product # 38-9100, 1-3 µg/mL) and detected by chemiluminescence Goat Anti-Rabbit IgG Secondary Antibody, HRP conjugate (Product # G-21234, 1:5000 dilution). A 130 kDa band corresponding to ZO-2 along with extra band was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE®10 % Bis-Tris gel (Product # NP0301BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane by over night wet transfer method. The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of (A) dog MDCK, (B) human Caco-2, (C) rat KNRK, and (D) mouse TCMK-1 cell lysates using Zymed (R) Rb anti-ZO-2 (C-term) (Product # 38-9100).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of Zona Occludens protein 2 (ZO-2) was performed by loading 50 µg of MDCK (lane 2) and NRK (lane 3) cell lysates and 2 µL SeeBlue® Plus2 Prestained Protein Ladder (Product # LC5925) (lane 1) per well onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane and blocked with 1% BSA/TBST for at least 1 hour at room temperature. ZO-2 was detected using a polyclonal antibody (Product # 38-9100) at a concentration of 1 µg/mL in blocking buffer overnight at 4°C on a rocking platform, followed by a goat anti-rabbit IgG Alexa Fluor 680 conjugated secondary antibody (Product # A-21109) at a dilution of 1:10,000 for at least 1 hour. Fluorescent detection was performed using the Odyssey® CLx imaging system (Li-cor Biosciences). Images generated by Joell Solan in Paul Lampe Lab at Fred Hutchinson Cancer Research Center.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Indirect immunofluorescence staining of MDCK II cells demonstrating co-localization (bottom, in yellow) of ZO-2 (top, in green) and ZO-1 (middle, in red) using Zymed (R) Rb anti-ZO-2 (C-term) (Product # 38-9100). Nuclei are stained with DAPI (blue). Image courtesy of Jacey Bennis and Dr. James Anderson, University of North Carolina at Chapel Hill, NC.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of ZO-2/TJP2 was done on 90% confluent log phase MCF-7 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with ZO-2/TJP2 Rabbit Polyclonal Antibody (Product # 38-9100) at 2 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d is a merged image showing cell junction localization. Panel e is a no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent detection of Zo-2 in MDCK cells. Confluent monolayers were fixed in 50%methanol/50%Acetone, blocked for at least 30 minutes in 1% BSA then incubated 2 hours with a Zo-2 rabbit polyclonal antibody (Product # 38-9100) at 2.5 µg/mL, washed, then incubated 1 hour with Alexa Fluor 488 conjugated Donkey anti-Rabbit secondary antibody (Product # A-21206) at 1:2000 dilution. Cells were counterstained with DAPI (blue). Coverslips were mounted with Prolong Gold Antifade reagent (Product # P36930) and imaged at 40X. Images generated by Joell Solan in Paul Lampe Lab at the Fred Hutchinson cancer Research Center.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 3: CRISPR-mediated knockout of TOCA-1 does not alter levels or localization of tight or adherens junction proteins. (A) Sequencing of PCR products amplified from genomic MDCK cell DNA in a region around the site of the putative CRISPR-mediated showed knockout cells had a 5-base pair deletion upstream of the PAM site (red GG) relative to wild-type DNA, resulting in a frameshift in the coding sequence and a premature stop codon at amino acid 36. (B) Comparison of expression by immunofluorescence analysis demonstrates that ZO-1 signal is identical in control and knockout cells (left and right). Junctional TOCA-1 staining is evident only in control cells, although the polyclonal TOCA-1 antibody generates some background signal in knockout cells (middle). (C) Enlargement of control ZO-1 and TOCA-1 staining reveals strong junctional colocalization (yellow signal). (D) E-cadherin (Ecad) localization is similar in control (top) and knockout cell lines (bottom). (E) In contrast to clear colocalization of TOCA-1 with ZO-1, enlargement of merge image between E-cadherin and TOCA-1 reveals that most E-cadherin is distributed along the lateral membrane, with little of the yellow signal suggestive of colocalization that is evident in C. Immunoblot (F) and immunofluorescence (G) analyses used to compare expression and localization of TOCA-1 and tight junction proteins in control and TOCA-1-knockout cell lines. Cldn2, claudin-2; Ocln, occludin. Bar, 15 mum.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 Construction of TALENs and ZO-1 gene knockout in MDCK I and II cells. (A) TALEN binding sites in the ZO-1 gene. The left and right arms of TALEN targeting sites are indicated in blue and the spacer region is indicated in red. The initiation codon within the spacer region is highlighted. (B) Immunofluorescence microscopic analysis of ZO-1, ZO-2 and ZO-3 in MDCK II cells transfected with TALEN constructs for ZO-1 gene knockout. After transfection, cells were subcultured on filter inserts for 4 d before analysis. At the boundary of control and ZO-1 knockout cells, characteristic convex curves of cell-cell junctions are observed ( arrows ). (C) Immunofluorescence microscopic analysis of ZO-1, ZO-2 and ZO-3 in MDCK I cells transfected with TALEN constructs for ZO-1 gene knockout. Similar morphological changes of cell-cell junctions at the boundary of control and ZO-1 knockout cells were observed in MDCK I cells. Staining of ZO-3 was reduced in ZO-1 knockout cells ( arrowheads ). Scale bars, 10 um.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Knockout of ZO-2 or ZO-3 in MDCK II cells. (A) TALEN binding sites in ZO-2 and ZO-3 genes. The left and right arms of TALEN targeting sites are indicated in blue and the spacer region is indicated in red. The initiation codon within the spacer region is highlighted. (B) Immunofluorescence microscopic analysis of ZO proteins in MDCK II cells transfected with TALEN constructs for ZO-2 or ZO-3 gene knockout. The shape of cell-cell junctions was unchanged by ZO-2 knockout (upper panels) and ZO-3 knockout (lower panels). Scale bar, 10 um.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 Effects of ZO-1 knockout on the localization of ZO-2, ZO-3 and occludin in the stable ZO-1 knockout clones. (A) Immunofluorescence microscopic analysis and line scanning of ZO-1 and ZO-2 in ZO-1 knockout clones 1 (KO 1) and 3 (KO 3) co-cultured with control cells. ZO-2 fluorescent signal at TJs was reduced in KO 1 but was slightly increased in KO 3. (B) Immunofluorescence microscopic analysis and line scanning of ZO-1 and ZO-3. ZO-3 signal at TJs was reduced but was increased in the cytoplasm in both of ZO-1 knockout clones compared to co-cultured control cells. (C) Immunofluorescence microscopic analysis and line scanning of ZO-1 and occludin. Occludin signal at TJs was reduced in both of ZO-1 knockout clones compared to co-cultured control cells. Scale bar, 10 um. (D) Immunoblots of ZO-2, ZO-3 and occludin in control MDCK II cells and ZO-1 knockout clones. Similar expression levels of ZO-2, ZO-3 and occludin were observed in control cells and knockout clones.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Effects of ZO-1 knockout on the localization of ZO-2 and ZO-3. (A) Effects of ZO-1 knockout on the localization of ZO-2 in MDCK II cells. The images in Figure 1 were used for the analysis. Signal intensity of ZO-2 and ZO-1 on lines shown in confocal microscopic image ( arrows ) were analyzed. ZO-2 fluorescent signal at TJs was reduced but was increased in the cytoplasm of ZO-1 knockout cells. (B) Effects of ZO-1 knockout on the localization of ZO-3 in MDCK II cells. ZO-3 fluorescent signal at TJs was slightly reduced but was increased in the cytoplasm of ZO-1 knockout cells. (C) Effects of ZO-1 knockout on the localization of ZO-2 in MDCK I cells. ZO-2 fluorescent signal was not altered in ZO-1 knockout MDCK I cells. (D) Effects of ZO-1 knockout on the localization of ZO-3 in MDCK I cells. ZO-3 fluorescent signal at TJs was markedly reduced in ZO-1 knockout MDCK I cells. Scale bar, 10 um.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 XIST expression in GECs and XIST regulated BTB permeability, glioma angiogenesis, tight junction-related proteins and CXCR7 expression. ( a ) Relative XIST expression in ECs and GECs by real-time qPCR. Data represent mean+-s.d. ( n =5, each). * P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 miR-137 mediated the effects of XIST knockdown on GECs. ( a ) TEER assay to evaluate the effect of XIST and miR-137 on BTB integrity. ( b ) HRP flux assay to evaluate the effect of XIST and miR-137 on BTB permeability. ( c ) Cell Counting Kit-8 (CCK-8) assay to evaluate the effect of XIST and miR-137 on GECs proliferation. ( d ) Transwell assay to evaluate the effect of XIST and miR-137 on GECs migration. ( e ) Matrigel tube formation assay to evaluate the effect of XIST and miR-137 on GECs tube formation. ( f ) Western blot assay to evaluate the effect of XIST and miR-137 on tight junction-related proteins. ( g ) Western blot assay to evaluate the effect of XIST and miR-137 on CXCR7. Data represent mean+-s.d. ( n =5, each). * P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 miR-137 expression in GECs and miR-137 regulated BTB permeability, glioma angiogenesis, tight junction-related proteins and CXCR7 expression. ( a ) Relative miR-137 expression in ECs and GECs by real-time quantitative PCR. Data represent mean+-s.d. ( n =5, each). * P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Quercetin attenuated diquat-induced ROS accumulation and reduced protein abundance of tight junction proteins in IPEC-1 cells. IPEC-1 cells were treated as described in Figure 2 . ( A ) DCFH-DA-positive populations were detected by flow cytometry analysis. ( B ) Intracellular ROS levels were determined by fluorescence microscope (magnification x200). ( C ) Intracellular GSH levels were measured. ( D , E ) Protein abundance for ZO-1, ZO-2, ZO-3, occludin, claudin-1, claudin-3, and claudin-4 were determined and analyzed by the Western blot assay. beta-actin was used as the loading control. Representative experimental results were repeated in three independent experiments and values are means +- SEMs, n = 3. Means without a common letter are considered as a significant difference, p < 0.05.

Supportive validation

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Experimental details: Figure 7. Direct binding of the angulin-1 pbm to ZO-1 and its role in tTJ formation. (A) Lysates of angulin-1-KO cells were incubated with GST and GST fusion proteins of aa 409-575 and aa 409-570 of angulin-1 (GST-ang575 and GST-ang570, respectively) and subjected to GST pull-down assays. The lysates (input) and precipitates with GST, GST-ang575, and GST-ang570 were analyzed by Western blotting with anti-ZO-1 mAb, anti-ZO-2 pAb, and anti-ZO-3 pAb. The precipitates were also immunoblotted with anti-GST pAb. (B) Schematic diagram of the domain structure of mouse ZO-1 protein with 1,745 amino acids. ZO-1 contains three PDZ domains, an SH3 domain, a guanylate kinase domain (GUK), and an acidic domain (AD) in its N-terminal half. Four distinct portions of ZO-1 indicated as solid lines with amino acid numbers were produced as recombinant fusion proteins with MBP. (C) GST pull-down assays of MBP-N-ZO-1 fusion protein with GST-ang575. Input: crude lysate of Escherichia coli expressing MBP-N-ZO-1. (D) GST pull-down assays of MBP fusion proteins of PDZ1, PDZ2, and PDZ3 of ZO-1 (MBP-zPDZ1, MBP-zPDZ2, and MBP-zPDZ3, respectively) with GST-ang575. Input: each MBP fusion protein. In C and D, the samples were subjected to SDS-PAGE followed by Coomassie Brilliant Blue (CBB) staining. (E) Schematic diagram of a chimeric protein of angulin-1Deltapbm with full-length ZO-1 (angulin-1Deltapbm-ZO-1) and a chimeric protein of angulin-1Deltapbm with PDZ1 domain of ZO-1 (angulin-1Deltapbm-PDZ1). (F)

Supportive validation

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Experimental details: Figure S1. Localization of TJ and tTJ proteins at TCs. (A and B) Double-immunofluorescence staining of frozen mouse kidney sections containing proximal tubules with anti-tricellulin mAb and anti-claudin-2 pAb (A) and anti-angulin-1 mAb and anti-claudin-2 pAb (B). Tricellulin and angulin-1 showed dotlike staining at TCs with claudin-2 staining (arrows). (C-E) Double-immunofluorescence staining of MDCK II cells with anti-ZO-1 mAb and anti-ZO-2 pAb (C), anti-ZO-1 mAb and anti-ZO-3 pAb (D), and anti-JAM-A pAb and anti-claudin-2 mAb (E). Confocal sections in the apical region, including TJ markers and the lateral region, together with the corresponding Z-stack images along the white dotted lines are shown. Arrowheads indicate TCs. Bars: 10 um (top of A and B and C-E), 5 um (bottom of A and B).

Supportive validation

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Experimental details: Figure S4. Characterization of ZO-1/ZO-2-dKO cells expressing ZO-1 and its mutant. (A) Double-immunofluorescence staining of MDCK II cells and ZO-1/ZO-2-dKO cells with anti-angulin-1 pAb and anti-E-cadherin mAb. Arrowheads indicate TCs. Bar: 10 um. (B) Western blotting of lysates of MDCK II cells and two ZO-1/ZO-2-dKO clones with anti-angulin-1 pAb, anti-ZO-1 mAb, anti-ZO-2 pAb, or anti-alpha-tubulin mAb. (C) Western blotting of lysates of MDCK II cells, ZO-1/ZO-2-dKO cells, and ZO-1/ZO-2-dKO cells expressing ZO-1-GFP or a ZO-1-GFP mutant lacking PDZ2 domain (ZO-1DeltaPDZ2-GFP) with anti-ZO-1 mAb, anti-GFP pAb, or anti-alpha-tubulin mAb. (D) Double-immunofluorescence staining of ZO-1/ZO-2-dKO cells expressing ZO-1-GFP or ZO-1DeltaPDZ2-GFP with anti-GFP pAb and anti-angulin-1 pAb. Confocal sections in the apical region, including ZO-1 constructs and the lateral region, together with the corresponding Z-stack images along the white dotted lines are shown. Arrowheads indicate TCs. Bar: 10 um. (E) Pull-down assays of lysates of ZO-1/ZO-2-dKO cells expressing ZO-1-GFP or ZO-1DeltaPDZ2-GFP with GST-ang575. Bound proteins, input of lysates, and GST-ang575 used in the assay were analyzed by Western blotting with anti-GFP pAb and anti-GST pAb.

Supportive validation

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Experimental details: FIGURE 1: ZO-1 and -2 are effectively depleted in stable MDCK II Tet-Off cell lines. (A) Western blot of ZO-1, -2, and -3 polypeptides in control, dKD (lines 1-3), and in dKD cells expressing a Tet-inducible full-length ZO-1 rescue transgene (ZO1R). ZO-1 expression in dKD lines is ~2-3% of that observed in control cells, while ZO-2 expression is almost undetectable. Note that ZO-3, while not directly targeted in dKD cells, is also reduced by up to 50%. In ZO1R cell lines, induction of ZO1R (I) restores ZO-1 and ZO-3 expression to levels equivalent to those in control cells, while ZO-2 expression remains suppressed. U, uninduced; I, induced. (B) Immunocytochemistry of ZO-1, -2, and -3 in the control and dKD-3 cell line. There is a dramatic reduction in the staining of all three peptides at the AJC, and the outline of the AJC is much more trapezoidal in dKD cells relative to control cells. All images are 1-mum-thick, maximum-density projections of the AJC. Scale bar: 10 mum.

Supportive validation

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Experimental details: Figure 1. ZO-1/ZO-2 are required for apical junction localization of TJ proteins. (A-J) Immunofluorescence analyses of parental MDCK II cells (A-J) and ZO-1/ZO-2 dKO cells (A'-J'). (A and B) ZO-1 (A) and ZO-2 (B) expression was abolished in ZO-1/ZO-2 dKO cells. (C-J) TJ markers including ZO-3 (C), occludin (D), JAM-A (E), claudin-1 (F), claudin-2 (G), claudin-3 (H), claudin-4 (I), and claudin-7 (J) were not concentrated to the apical junctions in ZO-1/ZO-2 dKO cells, and JAM-A (E) and claudins (F-J) were diffusely localized along the lateral and apical plasma membrane. Occasional apical junction accumulation of occludin and claudins, but not JAM-A was observed (arrowheads), colocalizing with ZO-3 (z-sections in D and G). Graphs are quantitation of the fluorescence intensity and represent mean +- SD ( n = 2-9). *, P < 0.05; ***, P < 0.0005, compared by t test. Scale bar: 20 um. n.s., not significant.