The GFP-ML1Nx2 probe does not dissociate from the Rab5-positive membranes in response to depletion of PtdIns3P and PdIns(3,5)P2.
Images show a representative cell expressing the three indicated constructs before and 40 min after treatment with 1 μM rapamycin, which induces recruitment of FKBP-MTM1 to the FRB-Rab5-decorated membranes, thereby depleting PtdIns3P and PtdIns(3,5)P2. 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 15 cells from four independent experiments). No dissociation of the GFP-ML1Nx2 is observed despite robust depletion of FYVE-EEA1 within 2 min of rapamycin application. Scale bar = 15 μm.