Fig. 5. EGF-induced colocalization of EGFR and GPI-GFP. (A) Colocalization active EGFR with GPI-GFP. HER14 cells expressing GPI-GFP were incubated for 1 hour on ice with 8 nM EGF-Rhodamine (EGF-Rho) for 60 minutes, or mock medium (without EGF-Rho). After fixation and embedding, the average lifetime values ± s.e.m. (^***P<0.0001) of GPI-GFP were determined as described in Materials and Methods and are presented in the histogram on the right. (B) HER14 cells expressing GPI-GFP were incubated for 1 hour on ice with 100 nM EGc5-A594. Activation with 8 nM EGF was performed on ice for 10 minutes. After fixation and embedding, mean lifetime values ± s.e.m. (^***P<0.0001) of GPI-GFP were determined as described in Materials and Methods and are presented in the histogram. (C) HER14 cells expressing GPI-GFP were pre-incubated or not with 5 µg/ml nystatin for 30 minutes at 37°C, for 1 hour on ice in the absence or presence of 100 nM CTB-A594, and stimulated or not for 10 minutes with 8 nM EGF. After fixation and embedding, average lifetime values ± s.e.m. (^***P<0.0001) of GPI-GFP were determined as described in Materials and Methods and are presented in the histogram. (D) HER14 cells expressing GPI-GFP were incubated for 1 hour on ice in the absence or presence of 100 nM CTB-A594 or CTB-biotin as control. Activation with 8 nM EGF was performed on ice for 10 minutes. After fixation and embedding, mean lifetime values ± s.e.m. (^**P<0.001) of GPI-GFP were determined as described in Materials and Methods and are presented in the histogram. (E) EGF induced the recruitment of GPI-GFP into the detergent-free lipid raft fraction. HER14 cells expressing GPI-GFP were stimulated or not with 8 nM EGF for 10 minutes, and detergent-free lipid raft fractions were isolated as described in Materials and Methods. Presence of EGFR, GPI-GFP and MAP kinase was analyzed by western blotting, presence of GM1 was determined using a dot-spot assay with CTB-HRP.