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Fig. 1. (A) Images of the localization of eYFP-H-Ras in transiently transfected HEK293 cells. Confocal images of HEK293 cells transiently expressing eYFP-H-Ras(N17), eYFP-H-Ras(V12) and eYFP-H-Ras(wt). Images were taken 48 hours after transfection. Fusions between eYFP and the wild-type H-Ras as well as both mutants of H-Ras showed clear plasma membrane localization, indicating that membrane targeting was not impaired by the eYFP fusion to H-Ras. Scale bar: 10 µm. (B) Insulin-induced GTP loading of eYFP-H-Ras(wt). Active Ras was specifically pulled-down in an RBD assay, separated on a PAA-gel, blotted and detected by anti-Ras antibody (see Materials and Methods). 3T3-A14 cells were transiently transfected with Ras membrane anchor (10 C-terminal amino acids) fused to eYFP (mt), eYFP-H-Ras(wt), eYFP-H-Ras(V12), or eYFP-H-Ras(N17). Results are shown for unstimulated (–) and 5-minute insulin-stimulated (+) conditions. A clear increase of GTP loading was observed for wild-type eYFP-H-Ras ( $~$ 50 kDa) after stimulation, as well as for endogenous Ras ( $~$ 21 kDa). (C) Activation of extracellular regulated kinases (ERKs) in the presence of eYFP-H-Ras(wt). Results are shown for unstimulated (–) and 5-minute insulin-stimulated (+) conditions. To show that the fusion of eYFP to H-Ras(wt) did impair downstream signaling, the phosphorylation of the MAPK-kinases ERK1 (44 kDa) and ERK2 (42 kDa) was tested. Activation of Ras by insulin, preparation of the cell lysates, SDS-PAGE and blotting were performed as described in Materials and Methods. The left part of the immunoblot (anti-ERK) shows total ERK1 and ERK2 in cell lysates of 3T3-A14 cells transiently transfected with eYFP-H-Ras(wt). The phosphorylation of ERK1 and ERK2 upon insulin addition is shown on the right (anti-ERK-P). The phosphorylation of the amino acids Thr202 and Tyr204 of ERK1 and ERK2 was specifically detected using a phospho-p44/42-MAPK antibody (Cell Signaling Technology, Beverly, MA, USA).

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