Figure 4: Dependence of EGFR phosphorylation and oligomerization-related structural parameters on ligand concentration.

(a) Western blot measurement of wild type total EGFR auto-phosphorylation in CHO cells exposed to increasing concentrations of EGF. The monoclonal pan-phosphotyrosine antibody 4G10 was used in the measurements. Data points and standard deviations are derived from the average of three independent measurements (examples western blot images shown in Supplementary Fig. 6c). (b) Similar to Fig. 3e, estimate of the relative population of EGFR dimers. The estimates are based on the wild-type FLImP distributions at varying EGF concentrations (Fig. 3e and Supplementary Fig. 7). Errors are calculated as in Fig. 3. (c) Western blot measurements of phosphorylation of Tyr1173 and Tyr992 in CHO cells exposed to increasing EGF concentrations. Data points and error bars (s.d.) are derived from the average of four independent measurements (examples shown in Supplementary Fig. 6a,b). For the Tyr992 data, P values were calculated using Student’s t-test to determine whether measured phosphorylation at high EGF concentrations was significantly different from the maximum phosphorylation value measured at 100 nM EGF. P values are: 50 nM EGF, P=0.058; 200 nM EGF, P=0.173; 500 nM EGF, P=0.027; 1,000 nM EGF, P=0.014; 2,000 nM EGF, P=0.011; 5,000 nM EGF, P=0.009. (d) The DOCA between EGFR-bound EGF molecules and the membrane, derived from FRET measurements shown in Supplementary Fig. 8. DOCAs were obtained from 1,000 bootstrap data sets (that is, data sets resampled with replacement). The error bars are the standard deviations of the bootstrap means. Simulations of the tetramer (Fig. 2d and Supplementary Fig. 5c) and dimer⁹ (bottom left inset) predict a DOCA of ∼5 nm for oligomers and ∼7.5 nm for dimers.