FIGURE 9.

Transformation by EGFR Mut R1-6 and ER-retained EGFR Mut R1-6/L393H and their ligand-independent phosphorylation are EGFR kinase-dependent. A, inhibition of colony formation of NIH 3T3 cells stably expressing WT EGFR, EGFR Mut R1-6, or EGFR Mut R1-6/L393H (LH) by the EGFR kinase inhibitor gefitinib (2.5 μm). Data shown are the mean ± S.D. from three independent experiments normalized to WT EGFR + EGF. ****, p < 0.0001. B, quantification of phospho-Tyr-1068 EGFR detected in the whole cell lysates from serum-starved NIH 3T3 cells expressing EGFR Mut R1-6/L393H treated as shown (gefitinib = 5 μm). Data were averaged from four independent experiments. Error bars show S.E., **, p < 0.01; n.s., not significant. C, serum-starved NIH 3T3 cells stably expressing either WT EGFR, EGFR Mut R1-6, or EGFR Mut R1-6/L393H were treated without or with EGF as in Fig. 3. Lysates (equal protein content) were untreated (Un) or were treated with Endo H (EH) or with PNGase F (PNG) for 24 h before Western blot analysis using anti-phospho-Tyr-1068-EGFR and anti-actin antibodies.