VPS35 associates with prenylated N-Ras, but not H- or K-Ras4B in intact cells. BHK cells were transiently transfected with the indicated constructs. GFP-tagged proteins are indicated in green, and mRFP-tagged proteins in red. The next day, cells were serum starved for 2 h before fixation, and FRET between the two tagged proteins was measured by FLIM. (A) Images of GFP lifetime represented in pseudocolor. Bar, 30 µm. (B and C) FRET efficiency values relative to the value of a GFP-mRFP fusion protein. Values plotted are mean ± SEM, n = 3 (>30 cells examined for each condition). ****, P < 0.0001; ns, not significant (compared with GFP-VPS35 plus mRFP–N-Ras). WT, wild type.