Claudin-2 is constantly recycled in MDCK cells and this recycling is inhibited by YM201636.

<p>(A) MDCK cells treated with YM201636, but not DMSO controls, showed accumulation of internal claudin-2 which colocalised with the internal claudin-1 (arrows). Some claudin-2 remained at the junctions (arrowheads). Scale bars 10 µm. (B) A surface biotinylation assay was performed on control or YM201636 treated MDCK cells and a Western blot from a representative experiment is shown. (C) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028659#s2" target="_blank">Results</a> represented graphically show the mean from four independent experiments, error bars are SEM. Claudin-2 was found to be endocytosed (endocytosis 60 min) and recycled (shown by the reduction in the ‘Recycling 20 min’ lane compared to the ‘Degradation Control’ lane). Degradation would be shown by a reduction in the ‘Degradation Control’ compared to the ‘Endocytosis 60 min’ but the mean from four experiments showed there was no detectable degradation over this time scale (shown graphically in C). Addition of YM201636 inhibited claudin-2 recycling and caused accumulation of endocytosed proteins.