Fig. 3

Rac1 knockdown with siRNA blocks direct lysosomal transport of APP. a SN56 cells were transfected with HA-βAPP-CFP (not shown) and LAMP1-mRFP (red), and 400 nM siRNA against Rac1. Cells were surface labeled on ice for with 647-labeled Anti-HA antibodies for 30 min and allowed to internalize APP for 15 min at 37 °C. Dye-labeled negative control siRNA was included with these transfections to confirm that they were transfected with siRNA (not shown). The approximate outline of each cell is superimposed for clarity. Co-localized pixels are shown in white. Scale bar = 5 microns. b Co-localization of internalized APP and LAMP1 in the presence of Rac1 siRNA was quantitated in at least 38 cells from at drawn from at least 4 experiments using a one-way ANOVA with Tukey post-test * denotes p < 0.05. c SN56 cells were grown in 60 mm dishes and transfected with increasing amounts of siRNA directed against Rac1. Cells were lysed and western blotted with antibodies against Rac1, stripped and reprobed with antibodies against tubulin. d Western blots from at least 3 experiments were quantified using ImageJ, and normalized to tubulin expression in 4 experiments using a one-way ANOVA with Tukey post-test. * denotes p < 0.05