Fig. 2.

WNK1 depletion increases the activity of PI3KC3. (A) WNK1 was knocked down with 20 nM siRNA in U2OS cells. After 1 d, the cells were transfected with a GFP-2xFYVE domain-expressing plasmid. After 2 d, cells were placed in normal or starvation medium containing DMSO (Fig. S2) for 4 h and then analyzed by immunofluorescence. The images were opened in Imaris 8 (deconvoluted) or ImageJ; images opened in Imaris 8 were subjected to background subtraction (filter width 26.6 μm). Cells were categorized as having obvious GFP-2xFYVE puncta or with diffuse GFP-2xFYVE expression. At least 100 cells were selected per condition in each experiment. (Scale bars, 12 μm.) (B) Quantitation of the fraction of GFP-2xFYVE punctate cells in A. **P < 0.01.