Fig. 1.

Regulated expression of WNK4 in MDCK II tet-off cell lines. Clones showing doxycycline-regulated expression of WT and mutant WNK4 were produced as described in Methods. (a) Immunoblot analysis of HA-tagged WT-WNK4, WNK4-E559K, and WNK4-Q562E. For each construct, independent clones (1, 2, and 3) were cultured in the presence (+) or absence (-) of doxycycline. Samples were fractionated by SDS/PAGE, followed by immunoblotting for WNK4 with anti-HA antibody. HA-tagged WT and mutant WNK4 migrate as ≈150-kDa species. (b and c) Induction and localization of WT-WNK4 in MDCK II cells. dox, Doxycycline. Cells were cultured, stained for WNK4 with anti-HA antibody, and visualized by immunofluorescence microscopy (see Methods). WNK4 expression is low in the presence of doxycycline (b) and is induced in its absence (c). WNK4 localizes to points of cell-cell contact. WNK4-D318A, WNK4-Q562E, and WNK4-E559K each showed a similar staining pattern (data not shown). (d) Localization of ZO-1 and WNK4. Cells were induced and stained for ZO-1 (green) and WNK4 (red). comp, composite. Both proteins localize to cell-cell junctions. (e) As in d, WNK4 signal only. (f) As in d, ZO-1 signal only.