Effect of Iressa on phosphorylation of p44/42 MAPK by (A) Immunofluorescence and phosphorylation of p44/42 MAPK and MEK1/2 by (B) flow cytometry in MAM-1 co-cultures. MAM-1 were subcultured on coverslips or in 6-well plates and grown to ~95% confluence and then treated by replacing the conditioned media with fresh media that contained diluent (.001% DMSO, Control) or Iressa for 2 hours prior to fixing and evaluation as described in the methods. A. Immunofluorescent photomicrographs were taken with the indicated objectives of cells that were double labeled for phospho-p44/42 MAPK (Thr202/Tyr204) in green (FITC) and α-SMA in red (TRITC) and counterstained with DAPI (blue) to define the nuclei. B. Dose-response for phosphorylation of p44/42 MAPK and MEK1/2 in MAM-1 co-cultures by flow cytometry. MAM-1 were treated as described above and dual-labeled for ErbB-2 and the indicated phospho-specific antigen. Bars represent Mean channel fluorescent values for pp44/42 MAPK (Thr202/Tyr204) (Blue Bars) or pMEK1/2 (Ser217/221) (Red Bars) in ErbB2+ (Solid Bars) and ErbB2-(Shaded Bars) subpopulations.