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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.
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