Specific activation of MAPK
by ΔB-Raf:ER increases expression of
PEG-3. B1TD cells were infected with either a control recombinant
adenovirus (CMV) or a recombinant adenovirus to express ΔB-Raf:ER.
Twenty-four hours after infection, cells were treated with 200 nM
4-hydroxytamoxifen (TAM) and either vehicle control (VEH, DMSO)
or 50 µM PD98059. Six hours after treatment,
cells were washed with PBS and frozen. (A) Cells
were lysed and prepared for immune complex kinase assays to determine
MAPK activity, as described in Materials and Methods. Data are the
means of three separate experiments ± SEM.
(B) Cells were lysed in preparation for SDS–PAGE,
followed by immunoblotting to determine PEG-3 protein expression
levels. A representative experiment is shown n = 3.
Lane 1, Ad.vec; lane 2, Ad.vec + TAM;
lane 3, Ad.vec + TAM + PD98059;
lane 4, Ad.ΔB-Raf:ER; lane 5, Ad.ΔB-Raf:ER + TAM; lane 6, Ad.ΔB-Raf:ER + TAM + PD98059.