Invitrogen

MARVELD2 Polyclonal Antibody

Product Image Gallery

MARVELD2 Antibody in Immunocytochemistry (ICC/IF) — Immunofluorescence analysis of Marveld2/ Tricellulin was performed using 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 2% BSA for 1 hour at room temperature. The cells were labeled with MARVELD2/Tricellulin Rabbit Polyclonal Antibody (Product # 48-8400) 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) 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 represents the merged image showing cell junctional localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.

MARVELD2 Antibody in Western Blot (WB) — Western blot analysis was performed on whole cell extracts (30 µg lysate) of HCT 116 (Lane 1) and COLO 205 (Lane 2). The blots were probed with Anti-MARVELD2/Tricellulin Rabbit Polyclonal Antibody (Product # 48-8400, 1- 2 µg/mL) and detected by chemiluminescence Goat Anti-Rabbit IgG (H+L) Secondary Antibody, HRP conjugate (Product # G-21234, 1:5000 dilution). A 68 kDa band corresponding to MARVELD2/Tricellulin was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0341BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). 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).

MARVELD2 Antibody in Western Blot, Immunocytochemistry, Immunoprecipitation (WB, ICC/IF, IP) — Figure 4 Tricellulin silencing does not lead to apical domain displacement at tricellular junctions. ( a ) Confocal microscopy analysis of tricellulin distribution on the apical ( Apical , upper panels) or lateral ( Lateral , lower panels) side in control ( Caco2 shNT , left panels) and EpCAM-depleted ( Caco2 shEpCAM , right panels) cells. xy and xz views are presented. Scale bars, 5 mum. ( b ) 3D-SIM microscopy analysis of tricellulin localization in control ( Caco2 shNT ) and EpCAM-deprived (C aco2 shEpCAM ) cells. While tricellulin was focused at the most apical part of the tricellular junctions in control cells, it was instead concentrated at the bottom of aberrant tricellular junctional tubes in the absence of EpCAM (white arrows). Scale bars, 2 mum. ( c , d ) Confocal microscopy analysis of double immunostainings for E-cadherin (green) and tricellulin (red) ( c ), occludin (green) and tricellulin (red) ( d ), on the basal or apical sides in EpCAM-depleted ( Caco2 shEpCAM ) cells. xy and xz views are presented. Scale bars, 2 mum. ( e ) Western blot analysis of the level of tricellulin expression in control ( Caco2 shNT ) or EpCAM-deprived ( Caco2 shEpCAM #1 and Caco2 shEpCAM #2 ) clones. alpha-tubulin was used as loading control. ( f ) Statistical analysis of tricellulin amounts relative to alpha-tubulin amounts in control ( Caco2 shNT ) or EpCAM-deprived ( Caco2 shEpCAM #1 and Caco2 shEpCAM #2 ) clones. The analysis was performed based on three independent experiments.

MARVELD2 Antibody in Immunocytochemistry, Immunohistochemistry (ICC/IF, IHC) — FIGURE 2: The localization of some, but not all, TJ proteins to the AJC is attenuated in ZO dKD cell lines. (A-C) A 1-mum-thick, maximum-density projection of the apical domain, en face, acquired at the AJC (see Materials and Methods ). (A) Occludin and cingulin localization at the AJC is reduced, whereas JAM-A and tricellulin appear relatively normal in dKD cells relative to control cells. Claudin-4 staining (green) is included with tricellulin staining (red) to highlight tricellular junctions. (B) Claudin-1 and -2 staining at the AJC is reduced, whereas claudin-3 and -4 appear relatively normal in dKD cells. Note again the dramatic change in cell shape at the AJC in dKD cells. Scale bar: 10 mum.

MARVELD2 Antibody in Western Blot, Immunohistochemistry (WB, IHC) — Fig. 7. Expression of tricellulin in tight junctions was decreased in Ildr1 -/- mice. (A-F) Immunofluorescence of the organ of Corti from the middle turn of P28 mice using tricellulin and ZO-1 antibody. Arrows in B,C show the localization of tricellulin in the tricellular tight junctions. (G) Western blot analysis of tricellulin in the basilar membrane at P15 and P21 Ildr1 +/- and Ildr1 -/- mice. Gapdh was used as a loading control.

Product Details

Applications

Tested Dilution

Publications

Western Blot (WB)

1-3 µg/mL

View 8 publications 8 publications

Immunohistochemistry (IHC)

View 2 publications 2 publications

Immunocytochemistry (ICC/IF)

2-4 µg/mL

View 2 publications 2 publications

Flow Cytometry (Flow)

3-5 µg/1x10^6 cells

Immunoprecipitation (IP)

View 1 publication 1 publication

Product Specifications

Species Reactivity

Human, Mouse, Rat

Published species

Dog, Human, Mouse

Host/Isotype

Rabbit / IgG

Class

Polyclonal

Type

Antibody

Immunogen

Recombinant protein derived from the C-terminal region of the human Tricellulin protein (Accession# NP_001033692, Q8N4S9), which is identical to rhesus monkey and chimpanzee sequences, and 89% homologous to rat and mouse, 88% homologous to bovine and canine, and 86% homologous to horse sequence

Conjugate

Unconjugated

Form

Liquid

Concentration

0.25 mg/mL

Purification

Antigen affinity chromatography

Storage buffer

PBS, pH 7.4

Contains

0.1% sodium azide

Storage conditions

Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

RRID

AB_2533851

Product Specific Information

This antibody is specific for the Tricellulin/MARVELD-2 protein. On western blots, it identifies the target band at ~64 kDa. Reactivity has been confirmed with human Caco-2, dog MDCK, mouse IMCD-3 cells, and mouse kidney lysates by western blotting, and with mouse MTE7b cells by immunocytochemistry. Based on amino acid sequence homology, reactivity with Rhesus monkey, chimpanzee, rat, bovine, and horse is expected.

Target Information

Tight junctions form an important barrier of paracellular transport in epithelial cells. Sealing of two adjacent cells at bicellular tight junctions, a point where three adjacent cells are in contact with each other. Tricellulin is the first protein identified that specifically concentrates in tricellular tight junctions. This protein has four membrane spanning domains, similarly to claudins. Tricellulin expression is high in epithelium-derived tissues, such as small intestine, kidney and lung. Functional evidence for the role of tricellulin in tight junction formation comes from siRNA studies, where suppression of its expression leads to compromised epithelial barrier and tight junction formation. Loss of function in tricellulin mutants missing all or most of a conserved region in the cytosolic domain which binds to the cytosolic scaffolding protein ZO-1, indicate that interaction with other known tight junction proteins plays an important role for the function of tricellulin.

For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.