Figure 2: Autonomously activated EphA2 is a monomer.

(a) Expression level dependence of LIFEA2 fluorescence lifetime (τ). Upper images: representative fluorescence- and τ-images of LIFEA2 expressed in Cos-7 cells. Upper row: mCitrine donor counts from LIFEA2, lower: τ (ns; color-coding right). First column: pre-stimulation; second: 20 min post-stimulation with pre-clustered ephrinA1-Fc, 2 μg ml⁻¹. Lower graph: 2D histogram of τ versus integrated mCitrine fluorescence intensity (F) for individual cells (n=54 cells) pre- (dark grey circles) and 20 min post-stimulation (light grey circles). (b) Expression level dependence of EphA2-mCitrine anisotropy. Upper images: representative fluorescence- and anisotropy-images of EphA2-mCitrine expressed in Cos-7 cells. First column: anisotropy images (A) of EphA2-mCitrine (color-coding right), second: EphA2-mCitrine fluorescence, third: dSH2-mCherry fluorescence, fourth: merge of EphA2-mCitrine (green) and dSH2-mCherry (magenta) fluorescence (white arrow points at colocalization). Upper row: pre-stimulation, lower: 20 min post-stimulation with pre-clustered ephrinA1-Fc, 2 μg ml⁻¹. Lower graphs: left: 2D histogram of A versus F for individual cells (n=41 cells) pre- (dark grey circles) and 20 min post-stimulation (light grey circles), right box: anisotropy of cytoplasmic mCitrine measured in multiple cells (n=27 cells) (+), mean±s.e.m. (0.273±0.004). Right: 2D histogram of Pearson's correlation coefficient (PCC) for colocalization versus F for individual cells pre- (dark grey circles) and 20 min post-stimulation (light grey circles). Scale bars: 10 μm.