Figure 5

Wnt1-induced extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation depends on epidermal growth factor receptor (EGFR) activity. (a) EGFR was immunoprecipitated from 2 mg of whole cell lysate from T47D/Wnt1, T47D/vector, SkBr3/Wnt1, and SkBr3 vector cells and analyzed by SDS-PAGE/immunoblotting for p-Tyr and EGFR. The p-Tyr signal corresponding to EGFR was quantified using the ImageQuant software. (b) T47D/Wnt1 cells were pre-treated for 1 hour with monoclonal antibody 225 containing conditioned medium (CM), and lysates were analyzed by SDS-PAGE/immunoblotting for p-ERK1/2 and ERK1/2. (c) T47D/Wnt1 cells were pre-treated for 1 hour with the metalloprotease inhibitor CGS27023A (50 μM) or dimethyl sulfoxide as control. Total lysates were analyzed by SDS-PAGE/immunoblotting for p-ERK1/2 and ERK1/2. (d) T47D cells were pre-treated for 1 hour with 2 μM PKI166 before treatment with Wnt1 CM or control CM. T47D/Wnt1 and SkBr3/Wnt1 cells were treated for 1 hour with 2 μM PKI166 prior to lysing the cells. Lysates were analyzed by SDS-PAGE/immunoblotting for p-ERK1/2 and ERK1/2. Tyr, tyrosine.