Figure 5: c-Cbl-mediated ubiquitination redirects ephrin-clustered EphA2 toward the LE. | Nature Communications

Figure 5: c-Cbl-mediated ubiquitination redirects ephrin-clustered EphA2 toward the LE.

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

Figure 5

(a) Time-lapse of EphA2 and Rab11a/Rab7 colocalization in Cos-7 cells on ephrinA1 stimulation. Left panels: fluorescence images of EphA2-mCitrine (left column), bfp–Rab11a (middle), mCherry-Rab7 (right), at indicated time in min. Right graphs: ratiometric analysis of EphA2-mCitrine (WT) and Y813F-EphA2-mCitrine (Y813F) localization on different membrane compartments, with and without ectopic expression of c-Cbl (Cbl). Upper: PM (PM-EphA2), middle: Rab11a-positive RE (RE-EphA2, inset: blow-up of the first 10 min), lower: Rab7-positive late endosome (LE-EphA2). Bar graphs: average amplitude between 48–53 min±s.e.m. (n=12–18 cells from at least three independent experiments). Upper right box: color-coding. (b) Immunostaining of endogenous EphA2, Rab7 and LAMP1 in Cos-7 cells before (left column) and 20 min post-stimulation of Cos-7 cells with pre-clustered ephrinA1-Fc, 2 μg ml−1 (right column). EphA2 (first row), Rab7 (second row), EphA2 (green) and Rab7 (magenta) overlay (third row), LAMP1 (fourth row), EphA2 (green) and LAMP1 (magenta) overlay (fifth row). Scale bars: 10 μm. (c) c-Cbl induces EphA2 ubiquitination and degradation. Left: western blot of Cos-7 total cell lysate (Total, upper) and anti-GFP IP EphA2-mCitrine or Y813F-EphA2-mCitrine (IP, lower) showing time course of stimulation with pre-clustered ephrinA1-Fc (2 μg ml−1). Pre-clustered Fc fragment (Fc, 2 μg ml−1) was used as control (dashed box). c-Cbl-bfp and HA-Ubiquitin were co-expressed. Total lysates were probed with anti-EphA2, anti-c-Cbl and anti-GAPDH antibodies. IP was probed with anti-HA for ubiquitin (Ub) and anti-EphA2 antibodies. Right graphs: upper, ratiometric quantification of EphA2/GAPDH from blots of EphA2 (red) or Y813F-EphA2 (green) (mean±s.e.m. normalized to zero time point, n=3 blots). Lower: ratiometric quantification of Ub (ubiquitin)/EphA2 from 3 blots of anti-GFP IP EphA2 (red) or Y813F-EphA2 (green), right boxes: control Fc fragment (120 min).

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