Fig. 4.

Phosphorylation of Y594/604 is reduced in EPHB1 mutants with compromised or reduced compartmentalisation phenotypes. Representative immunoblot analyses on total protein from DLD-1 cell lines expressing EPHB1 mutants with (A) wild-type-like (R865W, G685D and T117I), (B) compromised (C61Y and C183Y) and (C) reduced compartmentalisation phenotype (D762N, R351L, R90C and R170W) stimulated with 0.5 µM EphrinB1-Fc ligand or negative control Fc fragment. Phosphorylation of EPHB1 Tyr594/604 and total EPHB1 protein were detected with phospho-specific antibodies and FLAG-tag antibody, respectively. Phosphorylation was quantified as the ratio of band intensities of phosphorylated to total EPHB1 normalized to wild-type EPHB1 stimulated with ligand. Each experiment was repeated 3 times and the quantitation was based on results from all repetitions. Error bars, SD. The Mann–Whitney U test was used to calculate the P-values, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001