Figure 2: Constitutive EGFR signalling requires EGFR kinase activity but not ligand.

(a) Cetuximab fails to inhibit EGFR-mediated induction of IFIT1 mRNA as detected by real-time quantitative PCR. Cells were incubated overnight with either control IgG or Cetuximab (100 μg ml⁻¹). (b) Cetuximab effectively blocks EGF-induced tyrosine phosphorylation of EGFR. Cells were incubated overnight with Cetuximab or control IgG followed by exposure to EGF for 15 min followed by western blot with pEGFR antibody. (c) U251 cells conditionally expressing the mutant EGFRvIII in response to tetracycline were incubated with tetracycline (or no tetracycline) for 24 h followed by RNA extraction and quantitative PCR (qPCR) for IFIT1. (d) Western blot showing expression of EGFRvIII in U251 cells following tetracycline exposure. EGFRvIII is truncated and migrates faster on electrophoresis gels compared with endogenous EGFRwt. (e) U251 cells were transfected with EGFRwt, a kinase-inactive EGFR mutant, or empty vector followed by extraction of RNA and qPCR for IFIT1. (f) Expression of EGFRwt results in increased tyrosine phosphorylation of EGFR, which is further and substantially increased by EGF stimulation, whereas the EGFR kinase mutant fails to become tyrosine phosphorylated in response to EGF (an increase in tyrosine phosphorylation of the small amount of endogenous EGFRwt is seen). Cells were serum starved and cultured in serum-free DMEM overnight for all experiments. The EGF concentration used was 50 ng ml⁻¹. Error bars represent the means±standard deviations of three independent experiments. Data were analysed by one-way ANOVA followed by Tukey’s multiple comparison test. *P≤0.05, **P≤0.01, ***P≤0.001.