Figure 2

H-rasG12V nanoclustering largely depends on effector interactions, while Gal-1 interacts with Raf-effectors.

(A) Electron microscopic nanoclustering analysis of mGFP-H-rasG12V and mGFP-H-rasG12V-D38A with or without antisense-mediated knockdown of Gal-1 in BHK21 cells. Normalized univariate K-functions, where maximal L(r)-r values above the 99% CI for complete spatial randomness indicate clustering at that value of r (number of membrane sheets analysed per condition, n ≥ 10). (B) Complexation between indicated mGFP-tagged H-ras mutants and mRFP-tagged C-Raf-RBD or Gal-1 was determined using FLIM-FRET in HEK293-EBNA cells transiently expressing above constructs (two independent biological repeats). (C) Complexation between indicated EGFP-tagged full-length Raf proteins and mRFP-tagged Gal-1 measured by FLIM-FRET in HEK293-EBNA cells (three independent biological repeats). Examples of FLIM-FRET images of cells, coexpressing indicated FRET-pairs or EGFP-tagged C-Raf-RBD as donor-only control. Image colour look-up table on the right shows fluorescence lifetimes. (B,C) Plotted values correspond to the mean ± SEM. Numbers inside and above the bars indicate total number of cells imaged. The Methods section describes the indicated statistical comparisons (***p < 0.001). Samples with coexpressed fluorescent proteins mGFP and mRFP (B), or EGFP and mRFP (C) served as FRET controls. Note that non-control sample FRET-values were all significantly different from the (FRET-)control sample. (D) Analysis of the interaction between endogenous Raf isoforms and Gal-1 in BHK21 cells using in situ proximity ligation assay (PLA). Representative confocal microscopy images of indicated proteins are shown. The sample with siRNA-mediated Gal-1 depletion served as a negative control. Cell nuclei were stained with DAPI. Red foci indicate positive signals for protein interactions and their quantification is shown in the graph. Scale bar is 21 μm.