Figure 1.

Bex3 interacts with TrkA neurotrophin receptor. A, TrkA and Bex3 interact in HEK293-transfected cells. Lysates from HEK293 cells cotransfected with TrkA and HA-Bex3 were immunoprecipitated with Trk antibody (203) and Western blots were performed to detect the presence of Bex3 (HA) and Trk in the immunoprecipitates. The expression of TrkA and Bex3 in the lysates was detected with the corresponding antibodies. B, Endogenous Bex3 interacts with TrkA in PC12–6/15 cells. PC12–6/15 cell lysates were subjected to immunoprecipitation using Trk antibody (C-14) or rabbit-IgG as a control. Western blots were performed to detect the presence of Bex3 and Trk in the immunoprecipitates. C, Bex3 interacts with TrkA in PC12–6/15 cells in the presence and in the absence of NGF. PC12–6/15 cells were treated, or not, with NGF (100 ng/ml) for 15 min, and lysates were immunoprecipitated with Bex3 antibody (P-13). Western blots were performed to detect the presence of TrkA and Bex3 in the immunoprecipitates. The expression of TrkA and Bex3 in the lysates was detected with the corresponding antibodies. D, TrkA and Bex3 interact in rat DRGs. DRGs were dissected from E15.5 rat embryos and lysed for 2 h at 4°C. The lysates were immunoprecipitated with Trk antibody (203) or rabbit-IgG as a control, and Western blots were performed to detect the presence of Bex3 and Trk in the immunoprecipitates. E, The TrkA juxtamembrane domain participates in the interaction with Bex3. GST-TrkA recombinant proteins (left) were incubated with S³⁵-labeled Bex3 and resolved in SDS-PAGE. Autoradiography was performed to detect the interaction, and Coomassie staining was used to check GST fusion protein levels (right). F, Bex3 interacts exclusively with the juxtamembrane region of TrkA. Lysates from HEK293 cells cotransfected with GFP-Bex3 and TrkA or TrkA-ΔJM were immunoprecipitated with Trk antibody (203), and Western blots were performed to detect the presence of Bex3 (GFP) and TrkA (RTA) in the immunoprecipitates and lysates. Arrow indicates GFP-Bex3, and asterisks indicate a nonspecific band. G, The C terminus of Bex3 mediates its interaction with TrkA. TrkA and GFP-fused Bex3 expression plasmids (left) were cotransfected in HEK293 cells, and lysates were immunoprecipitated with Trk antibody (203). TrkA-interacting Bex3 was detected using GFP antibodies. Immunoprecipitated TrkA and Bex3 levels in the lysates were detected with the corresponding antibodies (right). Actin was used as a loading control.