LOK/SLK are the major ERM kinases. (A) Localization of endogenous LOK, ezrin, and actin in Jeg-3 wild-type and LOK^−/− SLK^−/−cells. Images are max projections except for yellow boxes, which represent magnification of apical Z-slices. (B) Ezrin and pERM levels in Jeg-3– or HeLa-knockout cells. Tubulin is used as a loading control. (C) Extracts of wild-type or LOK^−/− SLK^−/− cells were resolved by either 6% SDS-PAGE gel or 6% Mn-Phos-tag SDS-PAGE gel and blotted for ezrin and pERM. Approximately half of endogenous ezrin is phosphorylated in wild-type cells, as seen by the band shift. No phospho-shift is detected in LOK^−/− SLK^−/− cells. (D) Extracts of cells transfected with wild-type LOK or LOK mutants were collected and blotted for endogenous, untagged ezrin or pERM. Lysates were also blotted for Flag or LOK to check expression of the constructs relative to wild-type LOK. (E) LOK^−/− SLK^−/− cells were transfected with either LOK-GFP-Flag or K65R-LOK-GFP-Flag and then costained with ezrin and actin. K65R-LOK is unable to rescue apical ezrin localization. Vertical sections were expanded threefold for clarity. Scale bars, 10 µm unless otherwise noted in magnified sections.