YM201636 blocks the recycling of claudin-1.

<p>A surface biotinylation assay was performed on control or YM201636 treated MDCK cells. (A) The ‘Surface Biotinylated’ lane represents the initial biotinylated claudin-1 at the cell surface. Labelled claudin-1 that is internal after 60 min and is resistant to surface stripping is represented for control (‘Endocytosis 60 min’) and YM201636 treated (‘Endocytosis + YM201636) cells. Claudin-1 remaining internal (and thus not recycled back to the surface) following recycling for 20 min is represented as ‘Recycling 20 min’ for control and ‘Recycling + YM201636’ for treated cells. The recycled cargo is then determined as the reduction in signal in the recycling lanes compared to the endocytosis lanes. Addition of YM201636 blocked claudin-1 recycling and caused accumulation of endocytosed protein. Our previous study shows that degradation of claudin-1 does not occur over this timeframe <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028659#pone.0028659-Dukes1" target="_blank">[10]</a> so a block in degradation does not account for the accumulation of internal claudin-1. (B) <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 3 independent experiments, error bars are SEM.