Fig. 3. Cholesterol-dependence of the colocalization of GPI-GFP and EGFR with GM1. (A) Cholesterol-dependent colocalization of GPI-GFP with GM1. HER14 cells expressing GPI-GFP (donor) were incubated or not with 5 µg/ml nystatin for 30 minutes at 37°C and labeled or not with CTB-A594 (donor probe). The fluorescent intensity of GPI-GFP or CTB-A594 is shown in the left or middle panel, respectively. Fluorescent lifetime of GPI-GFP was analyzed as described in Materials and Methods and presented as false-colored images in the right panel. Histograms represent the mean lifetime values ± s.e.m. (^***P<0.0001) of GPI-GFP determined under the indicated conditions. (B,C) Cholesterol-independent colocalization EGFR with GM1 gangliosides. HER14 cells were treated with (B) 5 µg/ml nystatin or (C) 2.5 µg/ml filipin for 30 minutes at 37°C. Cells were labeled with anti-EGFR nanobody EGa1-A488 and labeled or not with the acceptor probe CTB-A594. Lifetime values were determined as indicated in Materials and Methods, and mean lifetime values of ± s.e.m. (^**P<0.001) are presented in histograms.