FIG 2.

EGFR mediates E4-ORF1-induced Erk1/2 activation. (A) The EGFR inhibitor AG1478 blocks EGF-induced EGFR autophosphorylation and Erk1/2 activation. Vector cells preincubated in minimal medium for 1 h were stimulated with EGF (5 ng/ml) mixed with either DMSO or AG1478 (1478) (0.5 μM) for 15 min, when cell extracts were prepared and immunoblotted for the indicated proteins. PY-EGFR detected with antiphosphotyrosine antibody PY20 comigrated with EGFR. (B) EGFR activity is required for E4-ORF1-induced Erk1/2 activation in MCF10A lines cultured in complete medium (CM). The indicated MCF10A lines cultured in complete medium were treated with DMSO or AG1478 (1478) (0.5 μM) for 1 h, when extracts were prepared and immunoblotted for the indicated proteins. (C) EGFR is also required for E4-ORF1-induced Erk1/2 activation in MCF10A cells incubated in serum and growth factor-depleted medium. wtORF1 cells cultured in complete medium were incubated in minimal medium (MM) containing either DMSO, LY294002 (LY) (100 μM), or AG1478 (0.5 μM) for 1 h, when cell extracts were prepared and immunoblotted for the indicated proteins. PY-EGFR was detected as described above for panel A.