Fig. 2. Nanoscale colocalization of EGFR with GM1 gangliosides. (A-C) HER14 cells grown on coverslips were incubated on ice with 100 nM (A) of the donor probes anti-EGFR nanobody EGa1 or (B) EGb4 directly conjugated to Alexa-Fluor-488, or with (C) 20 µg/ml transferrin-A488 (Tf-A488) in the absence or presence of 1 µg/ml of the acceptor probe CTB-A594. (D) HER14 cells expressing GPI-GFP were incubated or not for 1 hour on ice with 100 nM EGb4-A594 (acceptor). After fixation with 4% formaldehyde, average lifetime values of GFP were determined as described in Materials and Methods. Left panels represent the distributions of the donor probes with or without acceptor probe. The lifetimes are shown in the middle panels in false colors. Mean fluorescent lifetime values ± s.e.m. (^***P<0.0001) are presented in histograms on the right.