FIGURE 4.

MAPK pathway is investigated in chimera signaling. Before treatment, all cells were starved in serum-free medium for 24 h and then cultured in fresh assay medium. Then NIH-chimera cells were treated with 500 ng/ml AD5-10 or 500 ng/ml rsTRAIL for the indicated times. Lysates were probed for protein phosphorylation by Western blot using the respective specific antibodies. A, schematic diagram depicting DR5e/ErbB2i and derived chimeric receptors. B, expression of surface chimeras in NIH3T3 cells. NIH3T3 cells were transfected with chimeric constructs and stained with PE-labeled anti-DR5 antibody (FAB 71908) or AD5-10 and TRITC-labeled secondary antibody followed by flow cytometry. C and D, immunoblot analysis of the activation of ERK in NIH3T3 chimeras treated with 500 ng/ml AD5-10 (C) or rsTRAIL (D) for the indicated times. E and F, analyses of phosphorylated ERK1/2 and phosphorylated MEK1/2 levels in NIH3T3 wild-type or NIH3T3 chimera-transfected cells with 500 ng/ml AD5-10 (E) and/or rsTRAIL (F) treatment for 15 min. p-, phosphorylated; ICD, intracellular domain. All experiments were repeated at least three times with similar results.