Figure 1

Galectin-1 neither binds to a farnesylated Ras-peptide nor to H-ras in solution experiments.

(A,B) Fluorescence polarization binding assay of 0.25 μM fluorescein labelled and farnesylated Rheb peptide titrated with increasing concentrations of (A) the farnesyl-binding protein PDEδ or (B) purified Gal-1. (C) Sensitized acceptor FRET binding experiment of 250 nM H-ras and 250 nM Gal-1 or 250 nM C-Raf–RBD (RBD) fluorescently labelled using the ACP-tag technology. The legend to the left shows interaction partners schematically. H-ras was either GTPγS (GTP) or GDP loaded, as indicated. Fluorescent labelling substrates, coenzyme A (CoA)-linked ATTO-488 as a FRET-donor and DY-547 as FRET-acceptor, are represented by green and red stars, respectively. Control sample was a 1:1 mix of fluorescent labelling substrates each at 100 nM. Error bars indicate measurement error. (D,E) Interaction between H-ras and Gal-1 or the C-Raf-RBD (RBD) as indicated by the legend in (C) was determined by FLIM-FRET. Purified proteins as in (C) were incubated with fluorescent protein tagged proteins derived from BHK21 cell lysates (indicated with dotted cell outline). (D) Proteins from cell lysates were mRFP-tagged (red circle). (E) H-rasG12V labelled with mGFP (green circle) from lysates was used. Control is either mRFP-tagged C-Raf-RBD (upper column) or Gal-1 (lower column) incubated with 1 μM of CoA-488 in (D) and mGFP-H-rasG12V incubated with 1 μM of CoA-547 label in (E). (C–E) Binding of GTP-H-ras and the C-Raf-RBD served as a positive control. (D,E) Plotted values correspond to the mean ± SEM from three independent biological repeats. Numbers inside or above the bars indicate total number of imaged regions. The Methods section describes indicated statistical comparisons (ns, non-significant; ***p < 0.001).