Cigarette smoke activation of the EGFR is ligand-independent. Serum-starved HAE cells were incubated in DMEM with and without 40 nM 225 mAb on ice for 1 h followed by the indicated treatments in the absence or presence of 225 mAb at 37°C. NT, no treatment; EGF, 100 ng/ml for 15 min; GO, 1 U/ml for 30 min; CS, smoke from 1 cigarette for 45 min. EGFR was immunoprecipitated (IP) with anti-EGFR mAb 225; precipitated proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted (IB) with anti-phospho-Tyr (PY20) and anti-EGFR (RK2) Abs. p-EGFR, phospho-EGFR.