Figure 3.

The effect of surface-expressed ErbB-2 on the kinetics of ligand-induced tyrosine phosphorylation and MAPK activation. ErbB ligands were used to stimulate T47D breast cancer cells and their derivative, T47D-5R, which lacks surface expression of ErbB-2. A comparable number of cells was stimulated at 37°C by the indicated ligands (at 100 ng/ml) for various time intervals. Receptor activation, in whole cell lysates, was detected by immunoblotting (IB) with an antibody directed against phosphorylated tyrosine (P-TYR). MAPK activation in the same preparations was determined by using an antibody against the active doubly phosphorylated form of Erk proteins (Activated MAPK). For control of equal gel loading, the upper part of membranes used to detect MAPK was used to determine the amount of ErbB-2. Note that the 5R cells exhibited up-regulation of the cell-retained ErbB-2.