EphA3 and EphB2 coIP from cells and are coactivated by receptor-specific stimulation. (A) HEK293T cells transfected with diHcRed-EphA3 or GFP-EphB2 were stimulated for 15 min with ephrin-A5-Fc (A5-Fc), α-EphA3 mAb (IIIA4), or not stimulated (control [cont]). Ephrin or IIIA4-bound receptors were analyzed by a WB with the indicated antibodies. To control loading, lysates were blotted with α-EphA3 and α-EphB2 antibodies (bottom). Prot G p/down, protein G pull-down. (B) HEK293T cells transfected with EphA3 and/or with biotin ligase–treated AP-EphB2 were incubated with SA-coated Dynabeads for various times or with ephrin-B1-Fc/protein A Dynabeads as a control. Beads were recovered from cell lysates, and associated proteins were analyzed by a WB with α-EphA3 or α–PY-EphA3 antibodies. To control loading, total lysates were also blotted with α-EphA3 and α-EphB2 antibodies (bottom panels). (C) U251 glioma cells were treated with cross-linked IIIA4 and ephrin-B1-Fc to specifically activate EphA3 and EphB2, respectively, or left untreated (cont). (left) EphA3 IPs were immunoblotted for EphA3, PY, or associated EphB2. (right) Parallel EphB2 and EphA3 IPs were immunoblotted with α-EphA3, -EphB2, and -PY antibodies, as indicated.