Figure 1: EGF rapidly stimulates the ERK pathway in HT-29 cells. HT-29 cells were cultured in serum-free media overnight and stimulated with 50 ng/mL EGF or 10 ng/mL TNF (a). ERK activation in response to TNF is relatively delayed (apparent by 15 mins) relative to EGF (apparent by 5 mins). (b) shows an antiphosphotyrosine blot of immunoprecipitated EGFR following stimulation of serum-starved HT-29 cells with TNF-α. TNF-α treatment results in the time-dependent tyrosine phosphorylation of the EGF receptor. (c) shows the effect of EGF receptor tyrosine kinase inhibition using the EGFR tyrosine kinase inhibitor AG1478. Cells were treated for 15 mins with AG1478 (0–10 μM) and stimulated with 10 ng/mL TNF-α for 15 mins. AG1478 dose-dependently inhibits EGFR phosphorylation on tyrosine. Data are representative of at least three separate experiments.