Fig. 2.

Scatchard plots of EGF-binding data obtained from cells expressing EGFR-P (A) or versions of EGFR-P mutated in the autoinhibitory tether region, Del:560–590 (B) and D563A/H566A/K585A (C). Cells were grown to confluence in 24-well plates and incubated with increasing concentrations of ¹²⁵I-EGF in triplicate for 60 min at room temperature. A 100-fold excess of unlabeled EGF was simultaneously added to the third well to determine nonspecific binding. Curve fitting to saturation binding data (Insets) was performed by nonlinear regression using prism 3.03 software (GraphPad). EGFR-P cells express ≈600,000 EGFR per cell (A). The EGF-binding results best fit a linear Scatchard plot, although signs of convexity can already be observed. The convexity is much more pronounced in Scatchard plots of EGF-binding results to EGFR-P cells expressing 180,000 EGFRs per cell (see Fig. 7).