Figure 3
From: Galectin-1 dimers can scaffold Raf-effectors to increase H-ras nanoclustering
Galectin-1 directly binds to the Ras binding domain of effectors.
(A) Interaction of Gal-1 with fragments of C-Raf (as can be derived from Supplementary Fig. 2A) or with the RBD of PI3Kα studied by FLIM-FRET in BHK21 cells, transiently expressing mCit-tagged Gal-1 and mRFP-tagged RBD-constructs (three independent biological repeats). Fluorescence lifetimes of FRET-samples were all significantly different from the donor-control. 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 mRFP and mCit served as FRET controls. Note that non-control sample FRET-values were all significantly different from the (FRET-)control sample. (B) GST pull-down experiments were performed by mixing bacterially purified Gal-1 with GST, GST-C-Raf-RBD or GST-PI3Kα-RBD immobilized on glutathione sepharose beads. GST was used as a negative control. Proteins retained on the beads were resolved by SDS-PAGE and Western blotted using a monoclonal antibody (M01) against Gal-1 for detection. (C) Corrected sensitized acceptor emission FRET data of 100 nM ATTO-488-labelled Gal-1 titrated with increasing concentrations of DY-547-labelled C-Raf-RBD (scheme on the left). Both proteins were purified from bacteria and labelled with the ACP-tag method. The dissociation constant (Kd) was determined from the shown curve fit on the dataset of EmFRET as described in the Methods section. Plotted values correspond to the mean ± SEM.
