Figure 6: c-Cbl-mediated ubiquitination causes a transient EphA2 response. | Nature Communications

Figure 6: c-Cbl-mediated ubiquitination causes a transient EphA2 response.

From: Ubiquitination switches EphA2 vesicular traffic from a continuous safeguard to a finite signalling mode

Figure 6

(a) τ-maps of LIFEA2 and Y813F-LIFEA2 in Cos-7 cells on ephrinA1 stimulation (time; min). Upper panel: LIFEA2, lower: Y813F-LIFEA2. For each panel, upper row: mCitrine photon counts, lower: τ (ns; color-coding). Right: fluorescence images of c-Cbl-bfp. Right graph: normalized fluorescence lifetime; T=(τ–τ2/τ1–τ2)±s.e.m. of LIFEA2 (red) or Y813F-LIFEA2 (green), where τ1 is the initial and τ2 is the smallest fluorescence lifetime value. Data obtained from three independent experiments, n=7 cells. Scale bars: 10 μm. (b) Sustained downstream signalling of Y813F-EphA2-mCitrine (green) as compared with EphA2-mCitrine (red) in Cos-7 cells co-expressing c-Cbl-bfp on ephrinA1-Fc (2 μg ml−1) addition for the indicated time (min). In both cases, blots of total Cos-7 lysates were probed for phospho-Erk1/2 (pErk 1/2), total Erk1/2 (Erk1/2), phospho-Akt (S473) (pAkt) and total Akt (Akt). Right graphs: normalized ratio of phospho/total Erk1/2 (upper), or phospho/total Akt (lower)±s.e.m. from four independent blots. Color-coding above graphs (P values depicted above the compared time points; unpaired t-test). Pre-clustered Fc fragment (Fc, 2 μg ml−1) was used as control (dashed box).

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