![Figure 3. The interaction between PTP1B and EphA3 is kinase dependent. (A) The association between YFP-EphA3 and dHcRed-PTP1B-[D-A] in untreated or ephrinA5-stimulated COS7 cells was analyzed by confocal FLIM. Confocal micrographs of YFP-EphA3 (donor) fluorescence and the YFP fluorescence lifetime maps are shown. The boxed area in the bottom right is shown at higher magnification. (B) COS7 cells transfected with YFP-EphA3 and dHcRed-PTP1B-[D-A] were stimulated with ephrinA5, and live cells were analyzed by FLIM. The distribution of YFP fluorescence lifetimes at various time points during stimulation is plotted. The corresponding confocal images and lifetime maps are presented in Fig. S4 A. (C) NG108 cells, lacking endogenous Ephs (Elowe et al., 2001), cotransfected with cDNAs encoding GFP-PTP1B-[D-A] and w/t or mutant EphA3 (containing tyrosine-replacement or truncation mutants as indicated) were stimulated with ephrinA5 before lysis, α-EphA3 immunoprecipitation, and Western blot analysis with antibodies as indicated. The black line indicates that intervening lanes have been spliced out. Molecular mass standards are indicated next to the gel blots in kilodaltons.](https://cdn.rupress.org/rup/content_public/journal/jcb/191/6/10.1083_jcb.201005035/5/jcb_201005035_rgb_fig3.jpeg?Expires=1771638061&Signature=OhJ0o9in673dYCH0RCJx1vdclLlFUXI-PN2OlVegB1fKWFim6JQNTa8W2W~7t4GDfs3V3vyRPF6xjoA-PG1cqmOBA1f9WrLtmQIIv8sD9GzIOOi8pX1IS9YkxGQKNABJSZqqCDNauj73ek2pGSF3MCFoqYPC8phwIgO1wT1D4TeHF8cbyDyA9GBYTMm5d9EQkWrlVBNDuh0cByA0NUb91-rnKxHdZ11rz2sVLAitZWjw-svRmprBw3wAhcrMk2w4MdpB079DKSeCU~8GQxXGxX6y3g1PJ-Um4WSer4P84cPaZn5UZLvs2Qe89U~Q4RzrlUBPHG-DjeHyg0TKHJRD8w__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
The interaction between PTP1B and EphA3 is kinase dependent. (A) The association between YFP-EphA3 and dHcRed-PTP1B-[D-A] in untreated or ephrinA5-stimulated COS7 cells was analyzed by confocal FLIM. Confocal micrographs of YFP-EphA3 (donor) fluorescence and the YFP fluorescence lifetime maps are shown. The boxed area in the bottom right is shown at higher magnification. (B) COS7 cells transfected with YFP-EphA3 and dHcRed-PTP1B-[D-A] were stimulated with ephrinA5, and live cells were analyzed by FLIM. The distribution of YFP fluorescence lifetimes at various time points during stimulation is plotted. The corresponding confocal images and lifetime maps are presented in Fig. S4 A. (C) NG108 cells, lacking endogenous Ephs (Elowe et al., 2001), cotransfected with cDNAs encoding GFP-PTP1B-[D-A] and w/t or mutant EphA3 (containing tyrosine-replacement or truncation mutants as indicated) were stimulated with ephrinA5 before lysis, α-EphA3 immunoprecipitation, and Western blot analysis with antibodies as indicated. The black line indicates that intervening lanes have been spliced out. Molecular mass standards are indicated next to the gel blots in kilodaltons.
