Fig. 5. Interaction of AmphiTrk with mammalian neurotrophins. (A-D) Western blots of AmphiTrk induced phosphorylation of AKT and Erk1/2 following stimulation with mammalian neurotrophins in transfected cell cultures. Concentrations of neurotrophins are indicated above the blots (ng/ml) (E) Schematic representation of the chimaeric rat TrkA receptor containing the intracellular domain of AmphiTrk (HA-rTrkA-AmphiTrk). (F) Western blot of HA-rTrkA-AmphiTrk induced phosphorylation of AKT and Erk1/2 after stimulation with NGF. Detection of the HA epitope was carried out to confirm expression of the chimaeric receptor and the native rat TrkA, which was used as a positive control. (G) Detection of PLC phosphorylation after stimulation with NGF (100 ng/ml) and immunoprecipitation with anti-PLC. No phosphorylation of PLC was detected through HA-rTrkA-AmphiTrk. An anti-PLC antibody was used to control immunoprecipitation efficiency. Stimulation of cultures transfected with the empty vector (pCDNA3) was performed as a negative control in A-D,F,G. Tubulin detection was used to control the loading of the lanes in A-D,F,G. (H-K) Neurite outgrowth assays. Neurite outgrowth was induced by NGF stimulation and visualised by cotransfection with an enhanced yellow fluorescent protein (EYFP). (H) Empty vector (pCDNA3), (I) AmphiTrk, (J) chimaeric HA-rTrkA-AmphiTrk and (K) rTrkA. (L) Percentages of cells that developed neurites under the absence or presence of NGF stimulation.