Invitrogen

MARVELD2 Polyclonal Antibody

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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 was performed using Anti-MARVELD2 Polyclonal Antibody (Product # 48-8400) and 64, 62 kDa bands corresponding to MARVEL domain-containing protein 2 isoforms were observed across tested samples along with uncharacterized (*) bands at 45 and 80 kDa. Membrane enriched extracts (40 µg lysate) of HeLa (Lane 1), PANC-1 (Lane 2), MCF7 (Lane 3), U-2 OS (Lane 4), A-431 (Lane 5), HaCaT (Lane 6) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). 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 (0.25 µg/mL concentration) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036,1:20000 dilution using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Product # 34580). Higher expression in A-431 and HaCaT were observed compared to U-2 OS.

MARVELD2 Antibody in Western Blot, Immunocytochemistry (WB, ICC/IF) — Figure 9. Roles of tricellulin in tTJ formation and epithelial barrier function. (A) Lysates of four independent tricellulin-KO cell clones (tricellulin-KO_1-4) were analyzed by Western blotting with anti-tricellulin pAb or anti-alpha-tubulin mAb. (B) Tricellulin-KO_4 cells were immunostained with anti-tricellulin pAb and anti-E-cadherin mAb. Arrowheads indicate TCs. (C) Double-immunofluorescence staining of tricellulin-KO_4 cells with anti-angulin-1 pAb and anti-claudin-2 mAb or anti-angulin-1 pAb and anti-ZO-1 mAb. Arrowheads indicate TCs. Confocal sections of the apical region, including TJ markers and the lateral region, together with the corresponding Z-stack images along the white dotted lines are shown. The white arrow indicates an angulin-1-positive region at a TC without ZO-1. (D) Quantification of TCs with extended distribution of fluorescence signals of angulin-1, claudin-2, and ZO-1 to the basal direction. The data in MDCK II cells in the middle and right graphs are identical to those in Fig. 4 D . (E) Quantification of angulin-1 rods colocalizing with ZO-1 at TCs. (F) Paracellular flux of fluorescein in MDCK II and tricellulin-KO_1-4 cells. Data are shown as mean +- SD ( n = 3). P app , apparent permeability. (G) EM observation of horizontal ultrathin sections of tricellulin-KO_4 cells. TCs at the level of TJs (TJ level) and the more basal level of the lateral membrane containing desmosomes (DS level) were analyzed. The TJ level contained either the plasma membra

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 Immunohistochemistry (IHC) — Figure 5 JNK-pathway activity and cell polarity are not altered in the absence of MarvelD3. ( A ) Detection of JNK, c-Jun and their phosphorylated forms in protein extracts from 3 wild-type (+ / +) and 3 MarvelD3 knockout (-/-) pancreata by western blotting. No differences in JNK, phospho-JNK (P-JNK), c-Jun and phospho-c-Jun (P-c-Jun, Ser63 and Ser73) protein levels were observed between wild-type and knockout pancreata. Quantification of the bands (P-JNK/JNK and P-c-Jun/c-Jun) is shown on the right. Full-length blots/gels are presented in Supplementary Fig. 8. ( B ) Cell polarity assessment by immunolabeling of apical (ezrin, red) and basal (laminin, green) markers of epithelial (E-Cadherin, white) cells on E15.5 embryonic pancreas sections. Wild-type (+ / +) and knockout (-/-) pancreatic epithelial cells are polarized. ( C ) RT-qPCR evaluation of tight junction and baso-lateral marker gene expression levels in embryonic (E15.5 and E17.5) pancreata of wild-type (+ / + , green circles), heterozygous (+ /-, orange squares) and MarvelD3 knockout (-/-, red triangles) mice. Transient downregulation of claudin-3 and -7 , and NaK-ATPase ( Atp1a ) is observed in E15.5 knockouts. ( D-E ) Immunolabeling of the TAMP occludin and tricellulin (green) in the wild-type and knockout E15.5 pancreatic epithelium (E-Cadherin, red). The absence of MarvelD3 does not trigger relocalization of the two other TAMPs.

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

48-8400

Applications	Tested Dilution	Publications
Western Blot (WB)	0.25-3 µg/mL	View 10 publications 10 publications
Immunohistochemistry (IHC)	-	View 3 publications 3 publications
Immunocytochemistry (ICC/IF)	2-4 µg/mL	View 4 publications 4 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 View immunogen
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.

Product References

Angulin-1 seals tricellular contacts independently of tricellulin and claudins.

The Journal of cell biology

Sugawara T,Furuse K,Otani T,Wakayama T,Furuse M

Published figure using MARVELD2 polyclonal antibody (Product # 48-8400) in Immunocytochemistry

Mon Sep 06 00:00:00 EDT 2021

Angulin-1 seals tricellular contacts independently of tricellulin and claudins.

The Journal of cell biology

Sugawara T,Furuse K,Otani T,Wakayama T,Furuse M

Published figure using MARVELD2 polyclonal antibody (Product # 48-8400) in Immunocytochemistry

Mon Sep 06 00:00:00 EDT 2021

Spatio-temporal expression pattern and role of the tight junction protein MarvelD3 in pancreas development and function.

Scientific reports

Heymans C,Delcorte O,Spourquet C,Villacorte-Tabelin M,Dupasquier S,Achouri Y,Mahibullah S,Lemoine P,Balda MS,Matter K,Pierreux CE

Published figure using MARVELD2 polyclonal antibody (Product # 48-8400) in Immunohistochemistry

Thu Jul 15 00:00:00 EDT 2021

Luminal polyethylene glycol solution delays the onset of preservation injury in the human intestine.

American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Søfteland JM,Bagge J,Padma AM,Casselbrant A,Zhu C,Wang Y,Hellström M,Olausson M,Oltean M

Published figure using MARVELD2 polyclonal antibody (Product # 48-8400) in Western Blot

Tue Jun 01 00:00:00 EDT 2021

Association of tricellulin expression with poor colorectal cancer prognosis and metastasis.

Oncology reports

Zhang JX,Qin MB,Ye Z,Peng P,Li SM,Song Q,Lin L,Liu SQ,Xie LH,Zhu Y,Huang JA

48-8400 was used in Western Blotting to highlight for the first time the significance of tricellulin in colorectal cancer development and progression.

Sun Nov 01 00:00:00 EDT 2020

Association of tricellulin expression with poor colorectal cancer prognosis and metastasis.

Oncology reports

Zhang JX,Qin MB,Ye Z,Peng P,Li SM,Song Q,Lin L,Liu SQ,Xie LH,Zhu Y,Huang JA

48-8400 was used in Western Blotting to highlight for the first time the significance of tricellulin in colorectal cancer development and progression.

Sun Nov 01 00:00:00 EDT 2020

Diesel exhaust particle exposure reduces expression of the epithelial tight junction protein Tricellulin.

Particle and fibre toxicology

Smyth T,Veazey J,Eliseeva S,Chalupa D,Elder A,Georas SN

48-8400 was used in Western Blotting to determine the effect of diesel exhaust exposure on airway epithelial barrier function and composition using in vitro and in vivo model systems.

Thu Oct 15 00:00:00 EDT 2020

Diesel exhaust particle exposure reduces expression of the epithelial tight junction protein Tricellulin.

Particle and fibre toxicology

Smyth T,Veazey J,Eliseeva S,Chalupa D,Elder A,Georas SN

48-8400 was used in Western Blotting to determine the effect of diesel exhaust exposure on airway epithelial barrier function and composition using in vitro and in vivo model systems.

Thu Oct 15 00:00:00 EDT 2020

Diesel exhaust particle exposure reduces expression of the epithelial tight junction protein Tricellulin.

Particle and fibre toxicology

Smyth T,Veazey J,Eliseeva S,Chalupa D,Elder A,Georas SN

48-8400 was used in Western Blotting to determine the effect of diesel exhaust exposure on airway epithelial barrier function and composition using in vitro and in vivo model systems.

Thu Oct 15 00:00:00 EDT 2020

The extracellular domain of angulin-1 and palmitoylation of its cytoplasmic region are required for angulin-1 assembly at tricellular contacts.

The Journal of biological chemistry

Oda Y,Sugawara T,Fukata Y,Izumi Y,Otani T,Higashi T,Fukata M,Furuse M

48-8400 was used in Western Blotting to examine the molecular mechanism for the assembly of angulin-1 at the tricellular contacts.

Fri Mar 27 00:00:00 EDT 2020

Claudins and JAM-A coordinately regulate tight junction formation and epithelial polarity.

The Journal of cell biology

Otani T,Nguyen TP,Tokuda S,Sugihara K,Sugawara T,Furuse K,Miura T,Ebnet K,Furuse M

48-8400 was used in Western Blotting to demonstrate that claudins and JAM-A coordinately regulate tight junctions (TJ) formation and epithelial polarity.

Mon Oct 07 00:00:00 EDT 2019

Downregulation of lipolysis-stimulated lipoprotein receptor promotes cell invasion via claudin-1-mediated matrix metalloproteinases in human endometrial cancer.

Oncology letters

Shimada H,Satohisa S,Kohno T,Konno T,Takano KI,Takahashi S,Hatakeyama T,Arimoto C,Saito T,Kojima T

Published figure using MARVELD2 polyclonal antibody (Product # 48-8400) in Western Blot

Fri Dec 01 00:00:00 EST 2017

Blood-spinal cord barrier breakdown and pericyte deficiency in peripheral neuropathy.

Annals of the New York Academy of Sciences

Sauer RS,Kirchner J,Yang S,Hu L,Leinders M,Sommer C,Brack A,Rittner HL

Published figure using MARVELD2 polyclonal antibody (Product # 48-8400) in Western Blot

Sun Oct 01 00:00:00 EDT 2017

Contractile forces at tricellular contacts modulate epithelial organization and monolayer integrity.

Nature communications

Salomon J,Gaston C,Magescas J,Duvauchelle B,Canioni D,Sengmanivong L,Mayeux A,Michaux G,Campeotto F,Lemale J,Viala J,Poirier F,Minc N,Schmitz J,Brousse N,Ladoux B,Goulet O,Delacour D

488400 was used in immunocytochemistry and western blot to show that the absence of EpCAM in enterocytes results in an aberrant apical domain

Fri Jan 13 00:00:00 EST 2017

Contractile forces at tricellular contacts modulate epithelial organization and monolayer integrity.

Nature communications

Salomon J,Gaston C,Magescas J,Duvauchelle B,Canioni D,Sengmanivong L,Mayeux A,Michaux G,Campeotto F,Lemale J,Viala J,Poirier F,Minc N,Schmitz J,Brousse N,Ladoux B,Goulet O,Delacour D

488400 was used in immunocytochemistry and western blot to show that the absence of EpCAM in enterocytes results in an aberrant apical domain

Fri Jan 13 00:00:00 EST 2017

Contractile forces at tricellular contacts modulate epithelial organization and monolayer integrity.

Nature communications

Salomon J,Gaston C,Magescas J,Duvauchelle B,Canioni D,Sengmanivong L,Mayeux A,Michaux G,Campeotto F,Lemale J,Viala J,Poirier F,Minc N,Schmitz J,Brousse N,Ladoux B,Goulet O,Delacour D