Supporting Figures

Files in this Data Supplement:

Supporting Figure 6
Supporting Figure 7

Fig. 6. Dissociation kinetics for EGFR-P and D563A/H566A/K585A. Cells were seeded on 24-well plates and grown to confluency for ~48 h. Duplicate wells were incubated with a single concentration of ¹²⁵I-EGF at 4°C for 2.5 h. Cells were then washed twice in cold DMEM containing 0.1% BSA, and the final wash was allowed to remain on the cells for varying times ranging from 2 min to 3 h. After the dissociation period, cells were washed in PBS and lysed in 0.5 M NaOH, and a portion of each sample was counted in the liquid scintillation counter. Binding data were analyzed by one-phase exponential decay equation using PRISM software.

Fig. 7. Scatchard analysis of EGF binding to wild-type EGFR (A) and EGFR-P (B). Cells expressing wild-type EGFR or EGFR-P 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. (Upper) Nonlinear curve fitting to saturation binding data was performed as described before using PRISM software (GraphPad). (Lower) Typical curvilinear Scatchard plot was obtained for wild-type EGFR while EGFR-P produced convex Scatchard curve as an indication of positive cooperativity and lost high-affinity binding receptor class.