Figure 5

A dimerization deficient Galectin-1 mutant loses its effect on Ras nanoclustering and signalling.

(A) Nanoclustering-FRET response of H-rasG12V in dependence of Gal-1 or its dimerization deficient mutant N-Gal-1 in HEK293-EBNA cells expressing mGFP-/mCherry-H-rasG12V. (B) RBD-recruitment FRET response of H-rasG12V in dependence of Gal-1 or its dimerization deficient mutant N-Gal-1 (mGFP-H-rasG12V and mRFP-C-Raf-RBD expressed in HEK293-EBNA) to assess effector translocation from the cytoplasm to active H-ras in plasma membrane nanoclusters. (C) Left, Western blot analysis of HEK293-EBNA lysates expressing mGFP-tagged H-ras and mRFP-tagged Gal-1 constructs as indicated. Serum-starved cells were stimulated with 100 ng/ml EGF for the indicated times. Total ERK and phospho-ERK (pERK) levels were then determined by immunoblotting. β-actin is the loading control. Right, Quantification of three independent repeats of Western blot data as shown on left. The pERK-signal was normalized to the total ERK-signal. (D) The nanoclustering-FRET response of H-rasG12V, N-rasG12V and K-rasG12V with increasing concentration of Gal-1. BHK21 cells were transiently co-transfected with mGFP-/and mCherry-tagged Ras constructs alone (1.0) or with antisense-Gal-1 (0.5) or non-labelled Gal-1 (3.4). The cellular total Gal-1 concentration relative to endogenous Gal-1 in control BHK21 cells ([Gal-1]rel.) is displayed to the left of the data. (E) The nanoclustering-FRET response of K-rasG12V. HEK293-EBNA cells transiently expressed mGFP-/mCherry-K-rasG12V and if indicated non-labelled Gal-1 or N-Gal-1. Note that in (D) the FRET-levels for K-rasG12V are lower than in (E), due to the higher Gal-1 level in BHK21 as compared to HEK293-EBNA cells (Supplementary Fig. 4E). (A,B,D,E) Plotted values correspond to the mean ± SEM of three independent biological experiments. Numbers inside the bars indicate total number of cells imaged. The Methods section describes the indicated statistical comparisons (ns, non significant; *p < 0.05; ***p < 0.001); comparisons in (D) were done against the 1.0 parent-control. Samples with coexpressed fluorescent proteins mGFP and mCherry (A,E) or mGFP and mRFP (B) served as a FRET control. Note that non-control sample FRET-values were all significantly different from the (FRET-)control sample.