Fig. 3.

Quantification of the subcellular distribution of EGFR (EGF–Rh and ¹²⁵I-EGF) and Grb2–YFP. (A) 3D images of cells incubated with 2 ng/ml EGF–Rh at 37°C for the indicated times. Two examples of the quantification of the surface (Surface) and endosomal fluorescence (Inside) of EGF–Rh and Grb2–YFP in individual cells are presented. The calculations were performed using mask segmentation-based method described in the Materials and Methods. A.l.u.f.i, arbitrary linear units of fluorescence intensity. (B) The cells were incubated with 2 ng/ml EGF–Rh at 37°C for the indicated times, and further incubated with mAb528 at 4°C for 1 hour followed by fixation. Fixed cells were then incubated with the secondary anti-mouse antibody conjugated with Cy5. Merged fluorescence images of Rhodamine (red), YFP (green) and Cy5 (cyan) of individual 3D confocal sections are presented in the top row, and the merged images of Rhodamine and YFP are presented in the bottom row. The percentage of Grb2–YFP associated with the plasma membrane (PM Grb2–YFP) of total Grb2–YFP associated with the plasma membrane and endosomes (total) is shown on the right. The data are mean values (± s.e.m.) from 8–44 cells per data point. (C) HeLa-Grb2–YFP cells were incubated with 2, 4 or 20 ng/ml ¹²⁵I-EGF for indicated times. The amount of surface and internalized radiolabeled EGF was determined as described in the Materials and Methods and presented as the number of ¹²⁵I-EGF molecules per cell. Data are compiled from two independent experiments, with two replicates each. Error bars indicate standard deviations of the mean.