Journal: Cell Communication and Signaling : CCS

Article Title: CK2-dependent phosphorylation of occludin regulates the interaction with ZO-proteins and tight junction integrity

doi: 10.1186/1478-811X-11-40

Figure Lengend Snippet: Impaired binding of ZO-2 to the phospho-mimetic Occ-T400E/T404E/S408E construct. A ) The indicated GST-Occ cytoplasmic tail fusion proteins were used to pull down HA-ZO-2 from transiently transfected MDCK C11 cells with GSH-agarose beads. Isolated protein complexes were analyzed by Western blotting with anti-HA antibody. Equal amounts of GST- fusion proteins were pulled down as detected with an anti-GST antibody in the lower panel. B ) Quantification of 5 independent experiments as shown in (A). C ) Purified GST-occludin C-terminal domain (GST-OccC) was prephosphorylated in vitro by purified CK2 and subsequently used to pull down FLAG-tagged ZO-2 from transiently transfected HEK-293 cells. Association of FLAG-ZO-2 was analyzed by Western blotting with the anti-FLAG M2 antibody. D ) Densitometric quantification of 4 experiments as shown in ( C ).

Article Snippet: For immunofluorescence microscopy 1 × 10 6 MDCK C11 cells per well were seeded on chamber slides coated with collagen (0.1 μg/μl; Biochrom) and incubated for 24 h at 37°C, 5% CO 2 .

Techniques: Binding Assay, Construct, Transfection, Isolation, Western Blot, Purification, In Vitro

Journal: Cell Communication and Signaling : CCS

Article Title: CK2-dependent phosphorylation of occludin regulates the interaction with ZO-proteins and tight junction integrity

doi: 10.1186/1478-811X-11-40

Figure Lengend Snippet: Localization of wildtype occludin-FLAG 3 and the corresponding T400/T404/S408 mutated constructs. MDCK C11 cells stably transfected with the indicated FLAG 3 -tagged occludin constructs were stained with anti-ZO-1 (red) and anti-FLAG M2 (green) antibodies and nuclei were stained with DAPI (clones: mock 3.1, wt 1.1, T400A/T404A/S408A 3.1, T400E/T404E/S408E 4.1). Representative images of the indicated clones are shown. Images were taken on a confocal laser-scanning microscope. The lower right panel represents a merged image of the other three images. Bar, 20 μM.

Article Snippet: For immunofluorescence microscopy 1 × 10 6 MDCK C11 cells per well were seeded on chamber slides coated with collagen (0.1 μg/μl; Biochrom) and incubated for 24 h at 37°C, 5% CO 2 .

Techniques: Construct, Stable Transfection, Transfection, Staining, Clone Assay, Laser-Scanning Microscopy

Journal: Cell Communication and Signaling : CCS

Article Title: CK2-dependent phosphorylation of occludin regulates the interaction with ZO-proteins and tight junction integrity

doi: 10.1186/1478-811X-11-40

Figure Lengend Snippet: Expression of TJ proteins in the stably transfected MDCK C11 cells. A ) Claudin-1, claudin-2, ZO-1 and ZO-2 expression is not affected by the stable transfection of the indicated occludin-FLAG 3 constructs as detected by Western blotting. β-Actin was used as a loading control. Analysis of two different clones for each construct is shown. B ) Transfection of the indicated occludin constructs does not affect cell proliferation as investigated by a XTT-assay. The graph summarizes the results of 4 experiments including two clones of each construct (mean values +/− SEM).

Article Snippet: For immunofluorescence microscopy 1 × 10 6 MDCK C11 cells per well were seeded on chamber slides coated with collagen (0.1 μg/μl; Biochrom) and incubated for 24 h at 37°C, 5% CO 2 .

Techniques: Expressing, Stable Transfection, Transfection, Construct, Western Blot, Control, Clone Assay, XTT Assay

Journal: Cell Communication and Signaling : CCS

Article Title: CK2-dependent phosphorylation of occludin regulates the interaction with ZO-proteins and tight junction integrity

doi: 10.1186/1478-811X-11-40

Figure Lengend Snippet: Phosphorylation of occludin T400/T404/S408 regulates assembly/disassembly of TJs in Ca 2+ -switch experiments. A ) Confocal images were taken at t = 0 min after removal of Ca 2+ . B ) After depletion of Ca 2+ the phospho-mimetic Occ-T400E/T404E/S408E protein is rapidly dissociated from the TJs along with a loss of ZO-1 tight junctional staining. Wildtype occludin and Occ-T400A/T404A/S408A did not differ in the kinetics of disassembly. C ) After re-addition of Ca 2+ wildtype occludin and Occ-T400A/T404A/S408A rapidly reassembled into TJs whereas formation of TJs in Occ-T400E/T404E/S408E-transfected MDCK C11 cells was significantly delayed. Confocal images were taken 20 min after addition of Ca 2+ . Bar, 10 μM.

Article Snippet: For immunofluorescence microscopy 1 × 10 6 MDCK C11 cells per well were seeded on chamber slides coated with collagen (0.1 μg/μl; Biochrom) and incubated for 24 h at 37°C, 5% CO 2 .

Techniques: Phospho-proteomics, Staining, Transfection

Journal: Cell Communication and Signaling : CCS

Article Title: CK2-dependent phosphorylation of occludin regulates the interaction with ZO-proteins and tight junction integrity

doi: 10.1186/1478-811X-11-40

Figure Lengend Snippet: Increase of paracellular resistance after CK2-dependent phosphorylation of occludin. Two-path impedance spectroscopy was applied to measure the two components of epithelial resistance (R epi ), paracellular resistance (R para , reflecting the pathway across the tight junctions) and transcellular resistance (R trans , reflecting the pathway across the cell membranes). Measurements were done in MDCK C11 cells, which were stably transfected with the indicated occludin-FLAG 3 constructs. In Occ-FLAG 3 -T400E/T404E/S408E-transfected cells a dramatic increase in R para was detectable compared to wildtype occludin and to Occ-FLAG 3 -T400A/T404A/S408A-transfected cells, while R trans was unchanged. Due to the about fourfold increase of R para , the overall epithelial resistance R epi was also increased. The figure shows combined results of 6 independent measurements on two clones of each construct. ** p < 0.01.

Article Snippet: For immunofluorescence microscopy 1 × 10 6 MDCK C11 cells per well were seeded on chamber slides coated with collagen (0.1 μg/μl; Biochrom) and incubated for 24 h at 37°C, 5% CO 2 .

Techniques: Phospho-proteomics, Impedance Spectroscopy, Stable Transfection, Transfection, Construct, Clone Assay