Effect of EPHB2/p.R745C mutation on autophosphorylation of EPHB2 and ephrin-induced clustering. RBL-2H3 cells transfected with WT and mutant EPHB2 constructs were plated on fibrinogen coverslips for 1 hour. (A) Overexpression and phosphorylation of EPHB2-WT and EPHB2/p.R745C (EPHB2-μ) variant after activation with Cvx (1 nM) was quantified by western blotting using anti-EPHB2 and EPHB2-pY594/Y604 antibodies. Results are representative of 3 independent experiments. (B) Clustering of EPHB2 was performed by adding ephrin B1-Fc dimeric ligand to transfected cells stimulated by Cvx (1 nM) and plated on fibrinogen-coated coverslips for 30 minutes. After various washes, binding of ephrin B1-Fc and the level of EPHB2-GFP for the corresponding cell were quantified by fluorescence microscopy (Nikon Eclipse E600). Total IFI from 3 independent experiments is presented as the mean ± SEM.