Figure 7.

Media from irradiated cells contains a soluble factor, which can activate both MAPK and JNK in nonirradiated cells, which is blocked by a neutralizing anti-TGFα antibody. EGFR-CD533 and EGFR-antisense cells (as indicated) were cultured as described in MATERIALS AND METHODS. In D, cells were treated with doxycycline to induce EGFR-CD533 as in MATERIALS AND METHODS. Cells were irradiated (2 Gy), and 0 and 120 min after irradiation the media were removed from these cells. The media were incubated with either neutralizing anti-TGFα antibody or control antibody as in MATERIALS AND METHODS. Antibody-incubated media were added to nonirradiated plates, and 5 min after addition the plates were aspirated and snap frozen. (A) MAPK activity in EGFR-CD533 cells was determined after immunoprecipitation as in MATERIALS AND METHODS; (B) MAPK activity in EGFR-antisense cells was determined after immunoprecipitation as in MATERIALS AND METHODS. (C) JNK activity in EGFR-antisense cells was determined after immunoprecipitation as in MATERIALS AND METHODS. (D) MAPK activity in EGFR-CD533 cells expressing EGFR-CD533 was determined after immunoprecipitation as in MATERIALS AND METHODS. MAPK activity data are shown as fold increases in ³²P incorporation into MBP substrate and are normalized to activity at time = 0 from the means ± SEM of three independent experiments. JNK activity data are shown as fold increases in ³²P incorporation into GST-c-Jun substrate and are normalized to activity at time = 0 from the means ± SEM of four independent experiments.