The GFP-ML1Nx2 probe does not dissociate from the LAMP1-positive membranes in response to blockade of PtdIns(3,5)P2 synthesis.
Images show a representative cell expressing the indicated constructs before and 3 h after treatment with 1 μM YM201636, which inhibits PIKfyve that synthesizes PtdIns(3,5)P2. The montage images at the bottom are from the boxed region above, and are separated by 10 min intervals. The graph at right shows mean fluorescence intensity at LAMP1-positive membranes normalized to the whole cell for the indicated construct (data are means ± s.e.m. of 29 cells from three independent experiments). No dissociation of the GFP-ML1Nx2 is observed despite extensive vacuolation of the cells in response to YM201636 treatment. Scale bar = 15 μm.