GEEC Endocytosis in Drosophila Figure 1. Fluid phase and GFP GPI probes mark a distinct endocytic route at early time points in S2R+ cells. (A) FITC- or TMR- dextran (Fdex, Rdex respectively) was pulsed along with: malelyated BSA or A568/A647 TfR (mBSA, TfR respectively); or aGFP (to mark internalized GFP GPI) for 30–60 s. Cells were fixed and imaged with wide field microscope at high resolution. Colocalization of different probes in combination with each other was then quantified. Bar graph represents mean6s.e.m. from at least 3 independent experiments with 20–50 cells for each probe combination in each experiment. (B) Rdex was co-pulsed with A647Tf for 30 s in cells expressing Clathrin light chain GFP (Clc GFP) which were fixed and imaged with TIRF microscopy. Bar graph represents the mean6s.e.m. percentage of Clc GFP spots that colocalized with TfR and Rdex endosomes (measured from .600 endsomes of each type from 13 cells). Asterix on all bar graphs mark the comparison population mean value and refer to those means that are significantly different from controls (P,0.05 by Student’s t-test). The outline in white was traced using the phase contrast image of the cells obtained in bright field. Bars in A = 5 mm, B = 2 mm, insets magnified 2x. doi:10.1371/journal.pone.0006768.g001 PLoS ONE | www.plosone.org 4 August 2009 | Volume 4 | Issue 8 | e6768