Figure 7: Downmodulation of EGFR levels shifts the ubiquitination dose–response curve.

(a) We varied each parameter of the EAM 10-fold and computed the sensitivity coefficient S for the EGFR ubiquitination threshold position x_T. Only parameters whose variation resulted in a sensitivity coefficient of at least 0.1 are reported. (b) EGFR knockdown (KD) in HeLa cells was achieved by transfection with an anti-EGFR siRNA oligo. Control cells were transfected with mismatched oligo. Cells were then treated for 2 min with EGF as indicated. IP and IB were as shown. Quantitation of EGFR ubiquitination is shown in c. (c) Comparison of model predictions and experimental assessment of the ubiquitination threshold in EGFR-KD (KD) and control (WT) HeLa cells. Simulations were performed with EAM (in the simulation of the EGFR-KD the decrease in EGFR levels was assumed to be 4.2-fold, as determined in ¹²⁵I-EGF saturation binding assays). EGFR ubiquitination is expressed, for each condition, as normalized to the maximum value obtained in that condition (Ub/Ub_MAX). Experimental data are reported as mean±s.d. from at least three independent experiments.