Figure 8: Advanced model for EGFR Ub and pY as a function of EGF concentration and EGFR number.

(a) Left, relative EGFR phosphorylation (pY/EGFR_T, black lines) and ubiquitination (Ub/EGFR_T, red lines) levels, as given by the EAM, for the indicated EGF concentrations. The grey area represents the physiological range of EGFR levels. Dashed lines indicate the maximal phosphorylation and ubiquitination. Data are normalized to the maximum phosphorylation/ubiquitination at 100 ng ml⁻¹ EGF. Red squares and black circles represent experimental measurements of EGFR Ub and pY, respectively, obtained by the ELISA-based assay in NIH-EGFR clones with increasing numbers of EGFRs (from lowest to highest: NIH-EGFR^phy , physiological EGFR; NIH-EGFR^m-ov, medium overexpression; NIH-EGFR^h-ov, high overexpression). Right, representative immunofluorescence images of EGFR surface levels in the indicated NIH-EGFR clones. Scale bar, 18 μm. Bottom, number of surface EGFRs per cell, as measured by ¹²⁵I-EGF saturation binding. (b) Left, relative EGFR ubiquitination (left, Ub/EGFR_T, red line) and phosphorylation (right, pY/EGFR_T, black line), as given by the EAM, for the indicated EGF concentration, normalized to the maximum. Squares represent experimental measurements of EGFR ubiquitination (left) or phosphorylation (right), obtained by the ELISA-based assay in the indicated NIH-EGFR clones (red) or cell lines with increasing EGFR number (black, see also Supplementary Fig. 9). (c) Relative EGFR ubiquitination levels (Ub/EGFR_T), as given by the EAM, at 100 ng ml⁻¹ of EGF under control (red line) or Cbl overexpressing (red dashed line) conditions (modelled as a × 100 increase in line with data in Fig. 4b). Data are normalized to the maximum ubiquitination in each condition. Red squares and circles represent experimental measurements of EGFR ubiquitination, obtained by the ELISA-based assay, in the NIH-EGFR clones (as in a) infected with a lentiviral vector driving inducible Cbl overexpression (Cbl^oe, red circles), or empty vector (Vector, red squares), treated with doxocycline. Right, verification of Cbl overexpression in the indicated NIH-EGFR clones infected with empty vector (−) or vector driving inducible Cbl overexpression (+); tubulin, loading control. Densitometry analysis revealed an ∼80–100-fold Cbl overexpression of Cbl. Experimental data in a–c are reported as mean±s.d. from at least three independent experiments.