Enforced, selective activation of the ERK/MAPK pathway overrides stromal inhibition of megakaryocytic differentiation (indicated by gpIIb up-regulation).

Cells analyzed consisted of either K562 parent cells or K5ΔRafER.5 cells engineered to express the estrogen-responsive Raf-1 kinase mutant ΔRafER. Cells were treated for 48 hours as indicated with either 25 nM TPA or 1 μM estradiol in the presence or absence of HESS-5 stromal cell monolayers. Whole cell lysates from the indicated samples were subjected to immunoblot analysis with the indicated antibodies. Membranes were stripped and reprobed to correlate gpIIb, phospho-ERK, and tubulin expression within each sample.