Fig. 4. EGF does not affect colocalization of EGFR with GM1 gangliosides (A) EGFR is activated at 4°C. HER14 cells were stimulated with 8 nM EGF for indicated times and temperatures. Cell lysates were prepared and analyzed by western blotting for the presence of phosphorylated tyrosine at position 1068 (pEGFR). Actin staining was used as loading control. (B) Colocalization of activated EGFR with GM1. HER14 cells were incubated on ice with 8 nM EGF-488 (donor) in the absence or presence of 1 µg/ml acceptor probe CTB-A594. Confocal images representing the distribution of EGF-A488 are shown in the left panel. The lifetime of EGF-A488 was determined as described in Materials and Methods and presented in false colors in the middle panel. Mean lifetime values ± s.e.m. (^***P<0.0001) were determined and are presented in the histogram on the right. (C) EGF does not alter colocalization of EGFR and GM1. HER14 cells were incubated with 100 nM EGb4-A488 and 1 µg/ml CTB-A594 for 1 hour on ice. Cells were then either treated with 8 nM EGF for 10 minutes or left untreated. After fixation and embedding, lifetime values of EGb4-A488 were analyzed as described in Materials and Methods and are presented as mean lifetime values ± s.e.m. (^**P<0.001, ^***P<0.0001) in the histogram on the right.