RFP-HA-DAT was transiently co-expressed with W63A (A, B) or R60A (C, D) YFP-HA-DAT mutants in PAE cells, and cells were imaged through 561 (RFP) and 515 (YFP) filter channels. Maximal projection images of 3D images are presented in (A) and (C). Insets show high magnification of the region marked by the white rectangle to demonstrate the enrichment of RFP-HA-DAT in distal filopodia regions as compared to DAT mutants. Scale bars, 10 μm.
(B) and (D), Quantification of RFP/YFP fluorescence ratios in filopodia and other parts of the cells (“cell body”, CB) in images presented in A and C. Graph bars represent mean values (+S.E.M.) of the RFP/YFP ratios from 30–35 filopodia or ROIs within CB. A.u.f.i., arbitrary units of fluorescence intensity.