Fig. 1. Schematic representation and immunodetection of fusion proteins. (A) Depicted are Rab5, and fragments of Rabaptin5 and EEA.1 proteins fused to CFP/YFP at the N- or C-terminus. The first and the last amino acid residues in Rabaptin 5 (R5RB) and EEA.1 (EEA.1sh) fragments are numbered according to the full-length sequences. The FYVE domain of EEA.1 is indicated. PAE/EGFR cells transiently expressing wild-type (wt), Q79L or S34N CFP-Rab5 mutants or YFP-EEA.1 and R5BD-YFP were lysed, and CFP/YFP-fusion proteins present in lysates were detected by western blotting with anti-Rab5 or anti-GFP antibodies, respectively. All fusion proteins generated in this work migrated on SDS-PAGE according to the predicted molecular masses. (B and C) PAE/EGFR cells transiently expressing R5BD-YFP alone (B) or together (C) with CFP-EEA.1sh were incubated with 1 ng/ml EGF-Rh for 10 minutes at 37°C and fixed. EGF-Rh, CFP and YFP were detected using Cy3, CFP and YFP filter channels. Insets in (C) show an enlargement of the outlined regions. Bars, 10 µm.

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