- 710222 - Invitrogen Antibodies CLDN8 antibody

Submitted references A human surfactant B deficiency air-liquid interface cell culture model suitable for gene therapy applications.

Munis AM, Hyde SC, Gill DR
Molecular therapy. Methods & clinical development 2021 Mar 12;20:237-246

Campylobacter jejuni Serine Protease HtrA Cleaves the Tight Junction Component Claudin-8.

Sharafutdinov I, Esmaeili DS, Harrer A, Tegtmeyer N, Sticht H, Backert S
Frontiers in cellular and infection microbiology 2020;10:590186

Helicobacter pylori Employs a Unique Basolateral Type IV Secretion Mechanism for CagA Delivery.

Tegtmeyer N, Wessler S, Necchi V, Rohde M, Harrer A, Rau TT, Asche CI, Boehm M, Loessner H, Figueiredo C, Naumann M, Palmisano R, Solcia E, Ricci V, Backert S
Cell host & microbe 2017 Oct 11;22(4):552-560.e5

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Claudin-8 was performed on 90% confluent log phase Caco-2 cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with Claudin-8 Recombinant Rabbit Polyclonal Antibody (Product # 710222) at a dilution of 1:400 in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor® 488 Goat anti-Rabbit IgG secondary antibody (Product # A-11008) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c is a merged image showing cell junction localization and panel d is a control without primary antibody. The images were captured using a Nikon microscope at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1 Infection of polarized Caco-2 epithelial cell monolayers by C. jejuni 81-176 results in the disruption of claudin-8 patterns. Epithelial cells were immunostained with alpha - claudin-8 (green) and alpha- C. jejuni (red) without infection (A) , or after infection with C. jejuni wild-type (wt) strain (B) , Delta htrA knockout mutant (C) , Delta htrA complemented with protease inactive SA mutant (D) or wt htrA (E) . Infection was performed for 12 h at an MOI of 100. DAPI staining (blue) was used for the nuclear DNA counterstaining. White arrows indicate claudin-8 redistribution to agglomerates.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2 Infection of polarized T84 epithelial cell monolayers by C. jejuni 81-176 leads to disruption of claudin-8 and occludin in the tight junctions. Epithelial cells were immunostained with alpha-occludin (red), alpha-claudin-8 (green) and alpha- C. jejuni (magenta) without infection (A) , or after infection with C. jejuni wild-type (wt) strain (B) , Delta htrA knockout mutant (C) , protease inactive SA mutant (D) or Delta htrA complemented with wt htrA (E) . White arrows indicate occludin and claudin-8 redistribution to protein agglomerates. White boxes contain enlarged parts of tight junctions from corresponding smaller white boxes.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3 Localization of occludin and claudin-8 proteins in tight junctions and cytoplasm of T84 cells. The mean florescence intensity of the proteins was assessed separately in the membrane (m)-associated tight junctions and cytoplasm (c), and was calculated from 10 T84 cells per sample (A) . Single cell analysis of representative T84 cells without or with infection by wt C. jejuni . Infection of T84 cells leads to the drop of fluorescence intensity of claudin-8 in tight junctions along with its appearance in the cytoplasm (B) . Corresponding fluorescence intensity plots for occludin and claudin-8 within single cells between two yellow arrows, as marked in panel (B) . When infected with wt C. jejuni , a strong fluorescence intensity can be found in the cell cytoplasm (C) . RFU, relative fluorescence units of membrane and cytoplasmic localization. ***p < 0.001, ****p < 0.0001, ns, non-significant.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4 HtrA cleaves claudin-8 during C. jejuni infection in vivo . (A) Immunoblotting of protein extracts from polarized Caco-2 cell monolayers infected with C. jejuni wild-type (wt) or Delta htrA mutant. The blots were stained with polyclonal alpha-claudin-8 antibodies recognizing the carboxy-terminus of the protein. The alpha-HtrA and alpha-GAPDH blots served as controls. Besides the full-length protein (~26 kDa), a cleaved carboxy-terminal fragment (~18 kDa) was visualized with alpha- claudin-8 antibodies upon infection with C. jejuni wt. (B) Proposed cleavage site position in the amino-terminus of claudin-8 for HtrA protease according to the size of protein cleavage products determined by SDS-PAGE marker proteins. (C) A model for claudin-8 cleavage in the first extracellular loop (ECL1) by HtrA protease.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5 HtrA cleaves claudin-8 in a cleavage assay in vitro . (A) Recombinant GST-tagged human claudin-8 was incubated with either purified rHtrA in vitro . Immunoblotting against alpha-claudin-8 showed cleavage of the full-length GST-tagged claudin-8 protein, when incubated with recombinant HtrA resulted in the appearance of an 18-kDa claudin-8 fragment. In addition, a cleaved 33-kDa fragment was visualized in the blot with alpha-GST antibodies. (B) Mapping of the GST-claudin-8 cleavage fragments. The full-length GST-tagged claudin-8 protein (51 kDa) is cleaved into a carboxy-terminal 18-kDa fragment of claudin-8 and a 33 kDa amino-terminal fragment containing the GST-tag and the remaining 8-kDa amino-terminal fragment of claudin-8.

710222

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