PTP1B is recruited to sites of Eph/ephrin contact. (A) COS7 cells overexpressing YFP-EphA3 and dHcRed-PTP1B-[D-A] or w/t dHcRed-PTP1B were co-cultured (20 min) with ephrinA5/HEK293T cells and analyzed by FLIM to monitor direct interactions between EphA3 and PTP1B. Confocal images and YFP-EphA3 lifetime maps are shown. White broken lines mark the boundaries of EphA3-expressing cells; orange dotted lines mark those of ephrinA5 cells. Yellow arrows indicate EphA3-associated PTP1B-[D-A] and FRET at PM interfaces with ephrinA5-expressing cells. Open arrows indicate accumulated EphA3 in w/t dHcRed-PTP1B–expressing cells at sites of contact with ephrinA5 cells. (B) Co-cultures (20 min) between YFP-EphA3 expressing COS7 cells and ephrinA5-expressing HEK/293T cells were fixed, permeabilized, and stained with Cy3.5-tagged α-PY antibody to monitor EphA3 phosphorylation by using FRET between YFP-EphA3 and the Cy3.5-α-PY antibody. Dotted lines are as described in A. Yellow arrows indicate EphA3-associated Cy3.5-α-PY antibody and FRET at sites of marked EphA3 phosphorylation.