Fig. 3
WDFY2 depends on PI3P for endosome localization. a Protein–lipid overlay assay using purified full-length WDFY2. WDFY2 binds with high selectivity to PtdIns3P. Shown is a representative plot from two experiments. b Deconvolved widefield image showing GFP-WDFY2 localization to endosomes. GFP-WDFY2-R315A, a mutation in the binding site for PtdIns3P, abolishes the localization to endosomes and the protein is cytosolic. Representative image from 10 cells per condition. Scale bar: 10 µm. c hTERT-RPE1 cells stably expressing GFP-WDFY2 was treated with SAR405, Wortmannin or DMSO (as a bleaching control) with a final concentration of 6 µM. Cells were imaged every 5 s for 15 min WDFY2 spots were quantified per time point (5 cells per condition). Shown are mean ± 95% CI. d Distribution of GFP-2xFYVEWDFY2 and mCherry-2xFYVEHRS on a tubulating endosome. A WDFY2-derived 2xFYVE probe shows a preference for tubulating membranes. The graph shows the normalized fluorescence intensity of GFP-2xFYVEWDFY2 and mCherry-2xFYVEHRS along the indicated line. Representative image from 50 observed endosomes. Scale bar: 1 µm. e Coomassie Brilliant Blue stained gel showing top fractions from liposome flotation assays using His-MPB-fused of 2xFYVEWDFY2 and 2xFYVEHRS with differently sized liposomes. Shown is one representative gel from three independent experiments. f Plots showing the relative binding of 2xFYVEWDFY2 and 2xFYVEHRS to differently sized liposomes. Data shown are derived from three experiments. Shown are mean ± S.E.M. Source data are provided as a Source Data file
