Immunodetection of TPO-induced activation of intracellular signaling molecules.

Western blot (WB) or immunoprecipitation (IP) followed by Western blot was used to determine TPO-stimulated activation of Stat-3, Stat-5, Akt, and Erk1/2 (p44/42) in primary megakaryocyte-lineage cells expressing wild-type or mutant Mpl. TPO-stimulated cells (+) are compared to nonstimulated cells (−) for each Mpl construct. Equal protein loading was verified by the use of antibodies that detect total (phosphorylated and nonphosphorylated) forms of each signaling molecule tested.