Figure 5.

Radiation-induced secondary activation of EGFR is dose dependent and is mediated by TGFα. EGFR-antisense cells were cultured and were treated 30 min before irradiation with either control antibody or neutralizing antibody as described in MATERIALS AND METHODS. Cells were exposed to increasing doses of radiation (2–20 Gy). (A and B) After radiation exposure, at the indicated times media were aspirated, and plates were snap frozen; (C) after radiation exposure (180 min), media were aspirated, and plates were snap frozen. Cells were lysed, and identical portions (∼100 μg) from each plate were used to immunoprecipitate EGFR, followed by SDS-PAGE and immunoblotting versus either total EGFR protein or phosphotyrosine-containing (active) EGFR (EGFR-phosphotyrosine) as indicated. A representative experiment is shown (n = 3). Exposure time, 40 s.