Fig. 5. Detection of Rab5-CAAX activity in the plasma membrane. (A) Schematic representation and immunodetection of fusion proteins. Depicted is CFP-Rab5 protein fused to the 20-residue CAAX module of K-Ras4B at the C-terminus (CFP-Rab5-CAAX). PAE/EGFR cells transiently expressing CFP-Rab5-CAAX or its mutants, Q79L and S34N, were lysed, and the CFP-fusion proteins present in lysates were detected by western blotting with anti-Rab5 antibodies. All fusion proteins migrated on SDS-PAGE according to predicted molecular masses. (B) CFPRab5(Q79L)-CAAX was co-expressed with R5BD-YFP or YFP-EEA.1sh in PAE/EGFR cells. YFP, CFP and FRET^C images were obtained as in Fig. 2. Calculated FRET images presented as pseudocolor intensity-modulated images (FRET^C/CFP) (see Materials and Methods). Insets show an enlargement of the outlined regions. Arrows point to the absence of YFP-EEA.1sh associated with CFP-Rab5(Q79L)-CAAX at the plasma membrane. A.l.u.f.i, arbitrary linear units of fluorescence intensity. Bars, 10 µm.