Figure 6.

Role of WNK1 in the production of PI4P by expressed PI4KIIIα. (A) Chromatogram showing PI4K activity in membranes prepared from HeLa cells transfected with vector or cDNA for PI4KIIIα. Std: PI4P standard. (B) Chromatogram showing PI4P production by membrane-associated recombinant PI4KIIIα from cells that were co-transfected with control oligonucleotide or WNK1 siRNA. Relative knockdown of WNK1 is shown in the Western blot. (C) Mean ± SEM of PI4P production from 5 separate experiments, each with triplicate samples, as shown in panel B. *, p < 0.01 control versus knockdown. (D) Abundance of PI4KIIIα in the post-nuclear supernatant (PNS), cytosol, and membrane fractions from control and WNK1 siRNA-transfected HeLa cells. A western blot at bottom shows relative WNK1 knockdown. (E) Mean ± SEM (5 separate experiments) of PI4KIIIα abundance in the post-nuclear supernatant (PNS), cytosol, and membrane fraction in control (blue) and WNK1 knockdown cell (red), shown relative to the control. *, p < 0.05 between indicated. (F) Whole-cell TRPC6 currents stimulated by WNK1_1–491 in cells co-transfected with control oligonucleotide or siRNA for human PI4KIIIα, with either empty vector or plasmid that encodes rat PI4KIIIα. (G) Peak WNK1-induced inward current density from panel F. *, p < 0.01 between indicated.