Journal: bioRxiv

Article Title: Effects of Alternative Splicing-Specific Knockdown of Tjp1 α+ by Rbm47 on Tight Junctions Assembly during Blastocyst Development

doi: 10.1101/2023.07.18.549609

Figure Lengend Snippet: (A) The position of both isoforms of Tjp1 (exon11/12; α+/α-), the α-domain (exon20/21) and two Tjp1 α-specific (exon 19/21; w/o α and alternatively spliced exon 27/28; α-) primers used for cDNA amplification of Tjp1 isoforms. Arrows mark the position and direction of primers used for cDNA synthesis. Transcription levels of Tjp1 α+/-, Tjp1 α variant (B), and Tjp1 α+/-, Tjp1 α-, Rbm47 (C). Transcription levels of Tjp1 α+/α-, Tjp1 α-, and Rbm47, shown as relative quantification (RQ). Error bars represent mean ± standard error.

Article Snippet: Primary antibodies against RBM47 (HPA006347, Sigma-Aldrich), TJP1 α+ (HP9044; Hycult Biotechnology, Uden, Netherlands) and TJP1 α-(HP9045; Hycult Biotechnology) were diluted and incubated with the embryos overnight at 4°C.

Techniques: Amplification, Variant Assay

Journal: bioRxiv

Article Title: Effects of Alternative Splicing-Specific Knockdown of Tjp1 α+ by Rbm47 on Tight Junctions Assembly during Blastocyst Development

doi: 10.1101/2023.07.18.549609

Figure Lengend Snippet: (A) Expression and localisation of Tjp1 α+ and Tjp1 α-in the Rbm47 KD blastocyst. (B) Gene expression patterns of Tjp1 variants from 8-cell to hatching blastocyst (C) Proximity ligation assay (PLA) showing the interaction between Tjp1(mainly Tjp1 α-) and Ocln in blastocysts. 8C(8-cell), 8c-cmp (compacted 8-cell), Mo (Morula), Lt-Mo (Late morula), E-Bl (early blastocyst), Ex-Bl (Expanding-Bl), H-Bl (Hatching blastocyst). Antibodies host: Rabbit (Rb), Guinea pig (GP), mouse (m).

Article Snippet: Primary antibodies against RBM47 (HPA006347, Sigma-Aldrich), TJP1 α+ (HP9044; Hycult Biotechnology, Uden, Netherlands) and TJP1 α-(HP9045; Hycult Biotechnology) were diluted and incubated with the embryos overnight at 4°C.

Techniques: Expressing, Proximity Ligation Assay

Journal: The Journal of Cell Biology

Article Title: The keratin-binding protein Albatross regulates polarization of epithelial cells

doi: 10.1083/jcb.200803133

Figure Lengend Snippet: Impaired AJC formation in Albatross knockdown cells. (A) Double staining for Albatross (red) and the undercoat proteins (green) for each AJC component: TJ, ZO-1; AJ, afadin; DS, desmoplakin. Top and bottom columns show projections of x-y planes and z sections, respectively. Albatross knockdown A549 (Albatross KD) cells lack accumulation of these proteins at the cell–cell borders except in regions where residual Albatross is present. (B) Cell–cell adhesive properties evaluated by a cell aggregation assay. In the differential interference contrast images, control cells show cell aggregation. With Albatross knockdown A549 (A1050 and A1160) cells, the aggregated cell population is reduced and free cells are increased. The percentages of single cells in total cells (mean ± SD) are: control, 36.1 ± 3.9; A1050, 52.4 ± 2.8; A1160 cells, 59.4 ± 10.2. n = 4 and P < 0.01. (C) Immunoelectron microscopy of A549 cells with anti-Albatross antibodies. Note that the cytoplasm in the vicinity of AJCs is labeled. TJ, AJ, and DS are indicated. Arrows indicate cell–cell contacts. (D) Quantitative data from C. (E) BC fraction and AJ fraction were immunostained for Albatross with the indicated AJC proteins, PKCζ or Par3. Note that Albatross is well colocalized with them. (F) Immunoblotting of fractions derived from mouse liver: homogenates (left), BC (middle), and AJ (right). Not only Albatross but also Par3 is enriched in line with the concentrations of the indicated AJC components. (G) Immunoprecipitation of A549 cells with anti-Albatross antibodies. Start and IP indicate starting lysates and immunoprecipitates with preimmune (Pre.) and anti-Albatross (αAlb.) antibodies, respectively. Note the Par3 precipitation with Albatross. Among AJC components, ZO-1 also coprecipitated. (H) Immunoprecipitation analysis with tagged Albatross and Par3. Start and IP indicate starting lysates and immunoprecipitates with anti-GFP antibodies, respectively. Left lanes show results for negative controls expressing GFP alone. Par3 was the most precipitated with GFP-Albatross among coexpressed myc-Par3, -Par6, and -PKCλ. Bars: (A) 10 μm; (B) 100 μm; (C) 0.1 μm; (E, BC) 13 μm; (E, AJ) 10 μm.

Article Snippet: The following primary antibodies were used: monoclonal mouse anti-keratin 8 (Ks 8.7; Progen Pharmaceuticals), monoclonal mouse anti-keratin 18 (CY-90; Sigma-Aldrich), polyclonal mouse anti-pan keratin (Sigma-Aldrich), polyclonal guinea pig anti-K8/18 (Progen Pharmaceuticals), polyclonal guinea pig anti–desmoplakin 1 (Progen Pharmaceuticals), monoclonal mouse anti–desmoplakin 1 and 2 (Progen Pharmaceuticals), monoclonal mouse anti–ZO-1 (1; BD Biosciences), monoclonal rat anti–ZO-1 (BM173; Acris Antibodies, GmbH), monoclonal rat anti–E-cadherin (ECCD-2; EMD), monoclonal mouse anti-neurofilaments, monoclonal rat anti–platelet/endothelial cell adhesion molecule (anti-PECAM; CD31; BD Biosciences), monoclonal mouse anti–α-tubulin (B-5-1-2; Sigma-Aldrich), monoclonal mouse anti–claudin-2 (12H12; Invitrogen), monoclonal mouse anti–desmocollin-2/3 (7G6; Invitrogen), monoclonal mouse anti–desmoglein 2 (10G11; Progen Pharmaceuticals), monoclonal mouse anti–nectin-1 (CK8; Invitrogen), monoclonal mouse anti–β-catenin (14; BD Biosciences), polyclonal rabbit anti-ezrin (Millipore), rabbit anti-Par3 polyclonal antibody (provided by S. Ohno, Yokohama City University, Yokohama, Kanagawa, Japan; Millipore), monoclonal mouse anti-occludin (OC-3F10; Invitrogen), monoclonal rat anti–nectin-2 (502–57; HyCult Biotechnology), polyclonal rabbit anti-GFP (Santa Cruz Biotechnology, Inc.), polyclonal rabbit anti-PKCζ (Santa Cruz Biotechnology, Inc.), and polyclonal rabbit anti–glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) conjugated to HRP (Abcam).

Techniques: Double Staining, Immuno-Electron Microscopy, Labeling, Western Blot, Derivative Assay, Immunoprecipitation, Expressing

Journal: The Journal of Cell Biology

Article Title: The keratin-binding protein Albatross regulates polarization of epithelial cells

doi: 10.1083/jcb.200803133

Figure Lengend Snippet: Functions of keratins and Albatross–Par3 complexes. (A–C) The amounts of Albatross protein and mRNA were analyzed in both keratin 8 and keratin 18 (K8/18)-introduced SW13 cells. As a control, an empty vector was transfected. As loading controls, α-tubulin and GAPDH were used. Two independent experiments were performed. (A) Immunoblotting. In transiently K8/18-introduced SW13 cells, the amount of Albatross protein is elevated, along with the amount of keratin 18. (B) With stable lines, the same results were obtained. (C) RT-PCR. In K8/18-introduced SW13 cells, the mRNA level of K18 is elevated, but not that of Albatross. β-actin is included as an internal control. (D) Double staining for K8/18 and the indicated proteins: Albatross, AJC components of ZO-1 and afadin, and Par3. (top) In control cells, K8/18 is absent and only limited amounts of Albatross are apparent at cell–cell junctions. In stably K8/18-introduced SW13 cells, Albatross is well localized in cell–cell junctions compared with control cells. (middle and bottom) ZO-1, afadin, and Par3 similarly accumulated at the cell–cell borders in stably K8/18-introduced SW13 cells. (E) Immunostaining of stably K8/18-introduced SW13 cells transfected with control or Albatross siRNA. Note that ZO-1, afadin, and Par3 are reduced at cell–cell borders with knockdown of Albatross. (F) A model for the regulation of AJC and lateral domains with the Albatross–Par3 complex and keratins. Albatross–Par3 complexes regulate the formation of AJC and maintain lateral membrane identity. However, Par3 without Albatross regulates apical structures. Keratins stabilize Albatross, promoting the formation of AJC. Knockdown effects are also indicated. Bars, 10 μm.

Article Snippet: The following primary antibodies were used: monoclonal mouse anti-keratin 8 (Ks 8.7; Progen Pharmaceuticals), monoclonal mouse anti-keratin 18 (CY-90; Sigma-Aldrich), polyclonal mouse anti-pan keratin (Sigma-Aldrich), polyclonal guinea pig anti-K8/18 (Progen Pharmaceuticals), polyclonal guinea pig anti–desmoplakin 1 (Progen Pharmaceuticals), monoclonal mouse anti–desmoplakin 1 and 2 (Progen Pharmaceuticals), monoclonal mouse anti–ZO-1 (1; BD Biosciences), monoclonal rat anti–ZO-1 (BM173; Acris Antibodies, GmbH), monoclonal rat anti–E-cadherin (ECCD-2; EMD), monoclonal mouse anti-neurofilaments, monoclonal rat anti–platelet/endothelial cell adhesion molecule (anti-PECAM; CD31; BD Biosciences), monoclonal mouse anti–α-tubulin (B-5-1-2; Sigma-Aldrich), monoclonal mouse anti–claudin-2 (12H12; Invitrogen), monoclonal mouse anti–desmocollin-2/3 (7G6; Invitrogen), monoclonal mouse anti–desmoglein 2 (10G11; Progen Pharmaceuticals), monoclonal mouse anti–nectin-1 (CK8; Invitrogen), monoclonal mouse anti–β-catenin (14; BD Biosciences), polyclonal rabbit anti-ezrin (Millipore), rabbit anti-Par3 polyclonal antibody (provided by S. Ohno, Yokohama City University, Yokohama, Kanagawa, Japan; Millipore), monoclonal mouse anti-occludin (OC-3F10; Invitrogen), monoclonal rat anti–nectin-2 (502–57; HyCult Biotechnology), polyclonal rabbit anti-GFP (Santa Cruz Biotechnology, Inc.), polyclonal rabbit anti-PKCζ (Santa Cruz Biotechnology, Inc.), and polyclonal rabbit anti–glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) conjugated to HRP (Abcam).

Techniques: Plasmid Preparation, Transfection, Western Blot, Reverse Transcription Polymerase Chain Reaction, Double Staining, Stable Transfection, Immunostaining

Journal:

Article Title: Adrenomedullin stabilizes the lymphatic endothelial barrier in vitro and in vivo

doi: 10.1016/j.peptides.2008.09.009

Figure Lengend Snippet: Stimulation of HMVEC-dLys with AM does not affect gene expression of junction components. Total RNA was isolated from HMVEC-dLys treated with 0.1% BSA or 100 nM AM and the gene expression of ZO-1, Claudin-5, Claudin-12, JAMC, and VE-cadherin were assessed relative to GAPDH. No statistical significant differences were found between the two groups suggesting the effect of AM on barrier function is independent to changes in gene expression of junction components. n>3 with each assay performed in duplicate.

Article Snippet: Primer sets for human ZO-1, Claudin-5, Claudin-12, and JAMC were purchased from Qiagen.

Techniques: Gene Expression, Isolation

Journal:

Article Title: Adrenomedullin stabilizes the lymphatic endothelial barrier in vitro and in vivo

doi: 10.1016/j.peptides.2008.09.009

Figure Lengend Snippet: Genetic loss of AM does not affect gene expression of junction components in vivo. Using magnetic bead purification, endothelial cells were isolated from AM−/− and wildtype littermate embryos at embryonic day 13.5. The gene expression of ZO-1, Claudin-5, Claudin-12, JAMC, and VE-cadherin were determined and normalized to their expression in whole embryo. Although AM−/− endothelial cells had somewhat diminished gene expression levels, there were no statistically significant differences compared to wildtype endothelial cells. n>3 with each assay performed in duplicate.

Article Snippet: Primer sets for human ZO-1, Claudin-5, Claudin-12, and JAMC were purchased from Qiagen.

Techniques: Gene Expression, In Vivo, Purification, Isolation, Expressing

Journal: Cellular and molecular life sciences : CMLS

Article Title: THE PDZ2 DOMAIN OF ZONULA OCCLUDENS -1 AND -2 IS A PHOSPHOINOSITIDE BINDING DOMAIN

doi: 10.1007/s00018-009-0156-6

Figure Lengend Snippet: Structure and lipid binding properties of ZO PDZ2 domains. (A) Schematic overview of the domain organisation of the ZO-proteins. (B) Lipid-overlay assay of different domains of ZO-1 (human) and ZO-2 (mouse). Left is a schematic view of a lipid blot membrane (PIP-strips™). GST and GST-PH-LL5α were used as negative and positive controls, respectively. (C) ELISA PtdInsP binding assay. Each well of a ‘PIP specificity’ microtiter plate (Echelon Biosciences) was overlaid with GST, GST ZO-1 PDZ2, GST ZO-2 PDZ2, and GST- PH-PLC-δ1 (positive control). Data represent means ± STDEV (n=3). A.U. = absorbance unit.

Article Snippet: RNAi — siRNAs specific for human ZO-2 were obtained from Eurogentec (Seraing, Belgium).

Techniques: Binding Assay, Overlay Assay, Enzyme-linked Immunosorbent Assay, Positive Control

Journal: Cellular and molecular life sciences : CMLS

Article Title: THE PDZ2 DOMAIN OF ZONULA OCCLUDENS -1 AND -2 IS A PHOSPHOINOSITIDE BINDING DOMAIN

doi: 10.1007/s00018-009-0156-6

Figure Lengend Snippet: Peptide binding properties of ZO PDZ2 domains and mutants therein. (A) ITC titration of WT ZO PDZ2 domains with the C-terminal peptide (20 amino acids) of connexin43. Corrected raw heat data (top panel) and a binding isotherm (bottom panel) generated by plotting the area under the peak against the molar ratio of the peptide added to the PDZ domain are shown. (B) Data from pull down experiments measuring the binding of GST-ZO1 PDZ2 WT and mutants (upper panels) or GST-ZO-2 PDZ WT and mutants (lower panels) to the C-terminal peptide of connexin43 covalently bound to cyanogen bromide (CNBr)-activated Sepharose. GST-ZO2 PDZ2 WT is also shown. The right panel shows the Coomassie staining of the same volume of aliquots from individual proteins.

Article Snippet: RNAi — siRNAs specific for human ZO-2 were obtained from Eurogentec (Seraing, Belgium).

Techniques: Binding Assay, Titration, Generated, Staining

Journal: Cellular and molecular life sciences : CMLS

Article Title: THE PDZ2 DOMAIN OF ZONULA OCCLUDENS -1 AND -2 IS A PHOSPHOINOSITIDE BINDING DOMAIN

doi: 10.1007/s00018-009-0156-6

Figure Lengend Snippet: Lipid and peptide binding to ZO PDZ2 domains are mutually exclusive. (A) ELISA peptide binding assay showing that PtdInsPs compete with the Cx43 peptide for interaction with the ZO2 PDZ2 domain. Each well of a microtiter plate was coated with 1.0 µg Cx43 peptide and incubated with 1 µM GST or GST-tagged ZO-2 PDZ2 WT with or without pre-incubation with different PtdInsPs and PS (negative control). Data shown are representative of duplicate assays, each performed in triplicate. (RCU= relative colorimetric units). (B) Results from a pull-down assay measuring the binding of 3 µg GST-ZO2 PDZ2 WT to the C-terminal peptide of Cx43 covalently bound to CNBr-activated Sepharose with or without preincubation with different phospholipid liposomes at the indicated concentrations.

Article Snippet: RNAi — siRNAs specific for human ZO-2 were obtained from Eurogentec (Seraing, Belgium).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation, Negative Control, Pull Down Assay

Journal: Cellular and molecular life sciences : CMLS

Article Title: THE PDZ2 DOMAIN OF ZONULA OCCLUDENS -1 AND -2 IS A PHOSPHOINOSITIDE BINDING DOMAIN

doi: 10.1007/s00018-009-0156-6

Figure Lengend Snippet: ZO-proteins co-localise with PtdIns(4,5)P2. (A) Localisation of endogenous ZO-1 and ZO-2 in MDCK cells. Notice that ZO-1 exhibits mainly plasma membrane localisation whereas ZO-2 shows nuclear staining and cytoplasmic/membrane staining. (B) Endogenous ZO-1 and ZO-2 mark out areas where GFP-PH-PLCδ is localised. Nuclear ZO-2 is less clear because of focus on the plasma membrane. (C) Staining of MDCK cells for ZO-1/ PtdIns(4,5)P2 and ZO-2/PtdIns(4,5)P2 with 2C11 antibody. (D–E) PtdIns(4,5)P2 staining reveals co-localisation with endogenous ZO-2 (D) and ZO-1 (E) in polarised MDA-MB 231 breast cancer cells. Scale bar = 10µm.

Article Snippet: RNAi — siRNAs specific for human ZO-2 were obtained from Eurogentec (Seraing, Belgium).

Techniques: Staining

Journal: Cellular and molecular life sciences : CMLS

Article Title: THE PDZ2 DOMAIN OF ZONULA OCCLUDENS -1 AND -2 IS A PHOSPHOINOSITIDE BINDING DOMAIN

doi: 10.1007/s00018-009-0156-6

Figure Lengend Snippet: Endogenous ZO-2 localises in nuclear speckles containing PtdIns(4,5)P2 that disperse upon ZO-2 expression knock down. (A) MCF7 cells treated with 1µg/ml actinomycin D and stained for PtdIns(4,5)P2 with 2C11 antibody. Scale bar = 10µm. (B) Western blot analysis of total cell extracts from MCF7 cells (left panels) or HeLa cells (right panels), treated with a control siRNA or two independent siRNAs targeting ZO-2. Actin is used as a loading control. (C) Images of MCF7 cells and HeLa cells transfected with a control siRNA or an siRNA targeting ZO-2 and treated with actinomycin D. Speckle intensity is reduced and speckles appear to have desintegrated. The nuclear PtdIns(4,5)P2 pattern was visualised using 2C11 antibody. Scale bar = 10 µm.

Article Snippet: RNAi — siRNAs specific for human ZO-2 were obtained from Eurogentec (Seraing, Belgium).

Techniques: Expressing, Staining, Western Blot, Transfection