ZO-2 Antibody (38-9100)

来自 : 发布时间：2024-05-20

Clicking the images or links will redirect you to a website hosted by BenchSci that provides third-party scientific content. Neither the content nor the BenchSci technology and processes for selection have been evaluated by us; we are providing them as-is and without warranty of any kind, including for use or application of the Thermo Fisher Scientific products presented.    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).   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.   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).   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.   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.   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.   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. {KO}   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    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    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    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    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.   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.   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.   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.   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.   Published figure using ZO-2 polyclonal antibody (Product # 38-9100) in Immunocytochemistry   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. CD45 (LCA, leukocyte common antigen) is a receptor-type protein tyrosine phosphatase (PTP) ubiquitously expressed in all nucleated hematopoietic cells, comprising approximately 10% of all surface proteins in lymphocytes. CD45 is absent on non-hematopoietic cell lines, normal and malignant, non-hematopoietic tissues. CD45 glycoprotein is crucial in lymphocyte development and antigen signaling, serving as an important regulator of Src-family kinases. CD45 protein exists as multiple isoforms as a result of alternative splicing, differ in their extracellular domains but share identical transmembrane and cytoplasmic domains. CD45RA is an isoform of the CD45 complex and has restricted expression between different subtypes of lymphoid cells. CD45 isoforms differ in their ability to translocate into the glycosphingolipid-enriched membrane domains and their expression depends on cell type and physiological state of the cell. CD45 has been shown to be an essential regulator of T- and B-cell antigen receptor signaling and suppresses JAK kinases to regulate cytokine receptor signaling. CD45 is also important in promoting cell survival by modulating integrin-mediated signal transduction pathway, DNA fragmentation during apoptosis and inhibition or upregulation of various immunological functions. 蛋白别名: Friedreich ataxia region gene X104 (tight junction protein ZO-2); Tight junction protein 2; Tight junction protein ZO-2; Tight junction protein ZO-2 (Zonula occludens 2 protein) (Zona occludens 2 protein) (Tight junction protein 2); zona occludens 2; Zona occludens protein 2; Zonula occludens protein 2 Host server : magellan-srch-3-prod-green:8080/10.253.228.100:8080. git-commit: 2cd8645d2fc6bfe4ccb4abfa14772b0a94f68e98 git-url: http://victoria.invitrogen.com:8333/magellan/core.git git-branch: origin/release/1.27.0-2021.08.32-1.0