Phosphorylation of occludin T400/T404/S408 regulates assembly/disassembly of TJs in Ca2+-switch experiments. A) Confocal images were taken at t = 0 min after removal of Ca2+. B) After depletion of Ca2+ 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 Ca2+ 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 Ca2+. Bar, 10 μM.