Fig. 3.

Dimerization dependency of EGFR insertion mutations. (A) Western blot analysis of the N-lobe dimerization-deficient mutation (L760R or L704N) and the C-lobe dimerization-deficient mutation (V948R or M952R) on Y1197 autophosphorylation for WT and mutant (D770_N771insG, N771_P772insN, and D770$<$GY) EGFR. All of the constructs used are tagged with GFP except for L760R and L704N, which are tagged with FLAG. Cotransfection of V948R and L760R is indicated by V948R$+$L760R. Co-transfection of M952R and L704N is indicated by M952R$+$L704N. Eight percent SDS/PAGE is used to resolve the cell lysate sample. Y1197 phosphorylation of V948R$+$L760R and M952R$+$L704N is also analyzed by 5% SDS/PAGE to separate GFP- and FLAG-tagged constructs (SI Appendix, Fig. S1 A and B). Densitometry analysis of three to five independent experiments is shown in SI Appendix, Fig. S2). (B) A schematic plot summarizing the observed differences in WT and mutant EGFR in the different dimerization-deficient mutant backgrounds.