Fig. 7
An Arf6-DN mutant blocks APP transport to the lysosome but not the early endosome. a SN56 cells were transfected with HA-βAPP-CFP (not shown), LAMP1-mRFP (red) and a dominant negative mutant of a GTPase fused to fluorescent protein (yellow or cyan) as indicated. Cells were then surface-labeled with 647-labeled anti-HA antibody (green) for 30 min on ice and then allowed to internalize APP for 15 min at 37 °C and fixed. Each panel shows a single cell with approximate its outline superimposed for clarity. Merged red and green channels are shown, with co-localized pixels overlayed in white. Cell outlines are overlaid in white. Scale bar = 5 microns. c Quantitation of APP internalization to the lysosome with values normalized to HA-βAPP-CFP/LAMP1-mRFP cells examined at the time of the experiment. Colocalization was determined from more than 37 cells drawn from at least 4 transfections, and analyzed by a one way ANOVA with Tukey Post-test. * denotes p < 0.05. b Mouse cortical neurons were transfected with APP695, and LAMP1-mRFP (red), with or without Arf6-DN. Cells were then surface-labeled with 647-labeled 6E10 (green) and then allowed to internalize. Merged red and green channels are shown. Co-localized pixels overlayed in white. Scale bar = 5 microns. d Quantitation of APP internalization to the lysosome normalized to lysosome with values normalized to APP695/LAMP1-mRFP transfected cells at the time of the experiment. Data is from at least 58 cells in each group drawn from 5 experiments using a one-way ANOVA with Tukey post-test. * denotes p < 0.05. e SN56 cells were transfected with HA-βAPP-CFP (not shown), rab5-mRFP (red) and a dominant negative mutant of a GTPase as indicated. Merged red and green channels are shown, with co-localized pixels overlayed in white. Scale bar = 5 microns. f Quantitation of APP internalization to the early endosome, with values normalized to HA-βAPP-CFP/rab5-mRFP cells examined at the time of the experiment. Data is from more the 30 cells drawn from at least 4 transfections using one-way ANOVA with Tukey post-test. * denotes p < 0.05