The GFP-ML1Nx2 probe does not dissociate from the Rab5-positive membranes in response to blockade of PtdIns3P synthesis.

Images show a representative cell expressing the three indicated constructs before and 1 h after treatment with 100 nM wortmannin, which inhibits the PI 3-kinase that synthesizes PtdIns3P, the substrate for PtdIns(3,5)P2 synthesis. The graph at right shows mean fluorescence intensity at Rab5-positive membranes normalized to the whole cell for the indicated construct (data are means ± s.e.m. of 12 cells from three independent experiments). No dissociation of the GFP-ML1Nx2 is observed despite robust depletion of FYVE-EEA1 within 15 min of wortmannin application. Scale bar = 15 μm.