JCS028753 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Colocalization of EGFR and TfR with GM1 gangliosides. (A,B) HER14 cells grown on coverslips were incubated on ice with (A) 100 nM EGa1-A488 and 1 µg/ml CTB-A594, or (B) with 20 µg/ml transferrin-A488 (Tf-A488) and 1 µg/ml CTB-A594. After labeling, cells were fixed with 4% formaldehyde, embedded in Mowiol and examined by wide-field fluorescence microscopy.

• Supplemental Figure S2 -

  Fig. S2. Colocalization of EGFR with GPI-GFP. HER14 cells expressing GPI-GFP were grown on coverslips, and incubated for 1 hour on ice with 100 nM EGb4-A594 or, after fixation with 4% formaldehyde, were permeabilized and incubated with antibody against GM130. After embedding, the cells were imaged by wide-field fluorescence microscopy.

• Supplemental Figure S3 -

  Fig. S3. Nystatin does not affect the distribution of GPI-GFP or GM1. HER14 cells expressing GPI-GFP were preincubated or not with 5 µg/ml nystatin for 30 minutes at 37°C. The cells were labeled with 1 µg/ml CTB-A594, fixed with 4% formaldehyde and examined by wide-field fluorescence microscopy.

• Supplemental Figure S4 -

  Fig. S4. Cholesterol sequestration does not affect EGFR activity, and distribution EGFR and GM1. (A) HER14 cells were treated or not with 5 µg/ml nystatin for 30 minutes at 37°C, and stimulated for the indicated times with 8 nM EGF. Cells were lysed and analyzed for the presence of phosphorylated tyrosine at position 1068 (pEGFR) by western blotting. Actin was used as loading control. (B) HER14 cells were grown on glass coverslips and treated or not with 5 µg/ml nystatin for 30 minutes at 37°C. Cells were labeled with 100 nM anti-EGFR nanobody EGa1-A488 and with 1 µg/ml CTB-A594, fixed with 4% formaldehyde and examined by wide-field fluorescence microscopy.

• Supplemental Figure S5 -

  Figure S5. Analysis of FRET/FLIM data. (A) Intensity images from the four time-gate images and the lifetime image of GPI-GFP expressed in a NIH 3T3 cell. (B) Intensity images and lifetime image of the same cell after thresholding. (C) Histograms of lifetime distributions of one cell from non-thresholded pixels. (D) Similar histograms from thresholded pixels. Note the absence of the autofluorescence peak. (E) Determination of the average τ_donor value per cell by Gaussian fitting of the thresholded data as described in Materials and Methods. (F) Plotting the normalized average donor lifetime τ/τ_donor ± s.e.m. for n&γτ;5 cells per measurement.