Loss of clustered Cdc42 localization causes apical membrane enlargement downstream of ATP8B1. (A) Localization of YFP-Cdc42, WT or D185, in polarized control- or ATP8B1-depleted W4 cells. (B) Quantification of apical enrichment of YFP-Cdc42 in polarized W4 cells. Quantified as the ratio of the mean signal intensity between apical and basal membrane localized YFP-Cdc42. *, P < 0.005; n.s., P > 0.05. (C) Quantification of apical membrane size in polarized W4 cells expressing YFP-Cdc42 WT or D185. *, P < 0.0002. (D) HA-Cdc42(WT) and HA-Cdc42(D185) binding profile to membrane-spotted phospholipids. (E) Western blot for HA tag of immunoprecipitation eluates that were added to the lipid-spotted membranes in D, demonstrating that equal amounts of HA-Cdc42 were allowed to bind. (F) Quantification of apical membrane size in control W4 cells or ATP8B1-depleted W4 cells expressing YFP, YFP-Cdc42(WT), or YFP-Cdc42-PH. *, P < 0.00001; n.s., P > 0.05. E.V., empty vector. (G) Representative images of polarized W4 cells, control or ATP8B1 depleted, expressing YFP, YFP-Cdc42(WT), or YFP-Cdc42-PH in combination with the actin marker Lifeact-Ruby. Bars, 5 µm.