![Figure 1. PTP1B controls ephrin-induced phosphorylation of EphA3. (A) α-EphA3 immunoprecipitates and whole cell lysates from GFP-PTP1B, GFP-PTP1B-[D-A], or control vector-transfected EphA3/HEK293T cells, treated with ephrinA5-Fc, were analyzed by Western blotting with antibodies as indicated. The ratio of phosphorylated EphA3/total EphA3 (n = 3) was estimated (densitometry); mean ± SE are shown (error bars). (B) α-EphA3 immunoprecipitates and cell lysates of PTP1B, PTP1B-[D-A], or control vector (pMT2)-transfected 22Rv1 prostate carcinoma cells, with or without 10 min of ephrinA5 stimulation, were immunoblotted with the appropriate antibodies. Molecular mass standards are indicated next to the gel blots in kilodaltons. (C) EphrinA5-induced EphA3 phosphorylation in α-EphA3 immunoprecipitates from EphA3/HEK293T cells transfected with EGFP (control) vector, GFP-PTP1B, GFP-LMW-PTP, or GFP-SHP2, was quantified by densitometry analysis of Western blots (n = 3); mean PY-EphA3 levels, normalized to the EGFP-transfected control, ±SE are shown (error bars; ***, P < 0.001). (D) EphrinA5-induced EphA3 phosphorylation was monitored in PTP1B w/t and in control vector-transfected COS7 cells by confocal FLIM using YFP-EphA3 as fluorescence donor and Cy3.5-labeled PY72 as an acceptor. The YFP fluorescence lifetime maps are illustrated together with confocal micrographs revealing YFP-EphA3, PY, and PTP1B, the latter detected with labeled α-PY (Cy3.5PY72) and α-PTP1B (Cy5FG6) antibodies. Endogenous PTP1B (top) is shown at a higher display setting compared with recombinant w/t PTP1B (bottom). (E) The relative fraction (α) of membrane-proximal activated EphA3 receptors in PTP1B-overexpressing and control cells was calculated pixel-by-pixel from the ratio of the amplitude of the short lifetime component and the sum of the two amplitudes (n = 40 cells); mean ± SE are shown (error bars; **, P < 0.005). (F) The correlation between EphA3-YFP lifetimes (averaged across the whole cell) and the PTP1B expression level (determined from the fluorescence intensity of Cy5-α-PTP1B staining) is illustrated, where increased lifetime indicates decreased EphA3 phosphorylation. Each data point represents measurements from an individual cell.](https://cdn.rupress.org/rup/content_public/journal/jcb/191/6/10.1083_jcb.201005035/5/jcb_201005035_rgb_fig1.jpeg?Expires=1771638101&Signature=47oxvhbL~LSi2WINJJeo2isvOWI7vGg07Oy~0i1fuy~Joj41ocXlpz5fr3kknxzlLSG3CCL17UjvJVnjr0pObWz7gYCq0cIzPtEbNMx3fITj8zT3fFaamy1y2R9642TQQe2nE4YPb6RomXU2lDzqorngjmdLWpNyV0fqkGgnBJ6zuFyvJaxbdd-FQXT1pyU1GOZ-GSln0x6NdBCS8l60sCuti5E2~AEPM8koxSJLdVit17j7KTO6mlnd-1cQ4MIlA4x-k-feG7gNzelivEmgWha3PNl4HEZ1aJ62fhzh2TSBrJx-6ippItiqdE4AHxxr~gSYLRj9AKAIzaMNn3VSag__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
PTP1B controls ephrin-induced phosphorylation of EphA3. (A) α-EphA3 immunoprecipitates and whole cell lysates from GFP-PTP1B, GFP-PTP1B-[D-A], or control vector-transfected EphA3/HEK293T cells, treated with ephrinA5-Fc, were analyzed by Western blotting with antibodies as indicated. The ratio of phosphorylated EphA3/total EphA3 (n = 3) was estimated (densitometry); mean ± SE are shown (error bars). (B) α-EphA3 immunoprecipitates and cell lysates of PTP1B, PTP1B-[D-A], or control vector (pMT2)-transfected 22Rv1 prostate carcinoma cells, with or without 10 min of ephrinA5 stimulation, were immunoblotted with the appropriate antibodies. Molecular mass standards are indicated next to the gel blots in kilodaltons. (C) EphrinA5-induced EphA3 phosphorylation in α-EphA3 immunoprecipitates from EphA3/HEK293T cells transfected with EGFP (control) vector, GFP-PTP1B, GFP-LMW-PTP, or GFP-SHP2, was quantified by densitometry analysis of Western blots (n = 3); mean PY-EphA3 levels, normalized to the EGFP-transfected control, ±SE are shown (error bars; ***, P < 0.001). (D) EphrinA5-induced EphA3 phosphorylation was monitored in PTP1B w/t and in control vector-transfected COS7 cells by confocal FLIM using YFP-EphA3 as fluorescence donor and Cy3.5-labeled PY72 as an acceptor. The YFP fluorescence lifetime maps are illustrated together with confocal micrographs revealing YFP-EphA3, PY, and PTP1B, the latter detected with labeled α-PY (Cy3.5PY72) and α-PTP1B (Cy5FG6) antibodies. Endogenous PTP1B (top) is shown at a higher display setting compared with recombinant w/t PTP1B (bottom). (E) The relative fraction (α) of membrane-proximal activated EphA3 receptors in PTP1B-overexpressing and control cells was calculated pixel-by-pixel from the ratio of the amplitude of the short lifetime component and the sum of the two amplitudes (n = 40 cells); mean ± SE are shown (error bars; **, P < 0.005). (F) The correlation between EphA3-YFP lifetimes (averaged across the whole cell) and the PTP1B expression level (determined from the fluorescence intensity of Cy5-α-PTP1B staining) is illustrated, where increased lifetime indicates decreased EphA3 phosphorylation. Each data point represents measurements from an individual cell.
