Fig. 6. FIF analysis of oligomerization for EphA2 kinase-SAM linker mutants.

a Representative images of HEK293T cells transiently transfected with EphA2 5A-EYFP or EphA2 5E-EYFP and stimulated with ephrinA1-Fc or unstimulated (no ligand). More fluorescent patches appear to be present in the cells expressing the EphA2 5E mutant and stimulated with ephrinA1-Fc compared to the other conditions. Scale bar = 10 μm. b Normalized FIF brightness distributions for the EphA2 5A (n = 134) and 5E (n = 129) mutants in the absence of ligand treatment. Data are compared to previously published monomer (LAT) and dimer (E-cad, E-cadherin) controls⁴⁷. The brightness distribution curves for EphA2 5A and 5E are between the two controls, suggesting that both mutants exist in a monomer-dimer equilibrium. c Normalized FIF distributions for the EphA2 5A (n = 122) and 5E (n = 128) mutants stimulated with a saturating concentration of ephrinA1-Fc. The brightness distribution curve for the EphA2 5A mutant is between the two control curves, whereas the EphA2 5E mutant distribution curve is shifted to larger brightness values than the E-cadherin curve, suggesting the formation of higher-order oligomers. d, e Comparison of the normalized FIF distributions for d the EphA2 5A mutant and e the EphA2 5E mutant with and without ephrinA1-Fc. EphrinA1-Fc causes only a small shift in the curves for the EphA2 5A mutant but a large shift in the curve for the EphA2 5E mutant, suggesting that the EphA2 5E mutant can much more readily form higher order oligomers. The counts of molecular brightness were normalized, averaged, and plotted along with their standard errors in b–e. Source data for b, c, d, e are provided in the Source Data file.