Tpo stimulates ubiquitination of c-Mpl. (A) BaF-Mpl cells were treated with or without MG-132 and rhTpo for 0 to 120 minutes and c-Mpl ubiquitination was analyzed by c-Mpl immunoprecipitation (IP) and immunoblot (IB) analysis using an anti-mono/polyubiquitin antibody. Ubiquitination is seen as a high-molecular-weight smear. The data shown are representative of 3 independent experiments. The equivalence of protein loading was analyzed by reprobing the IB with an anti–c-Mpl antibody. (B) Bone marrow–derived megakaryocytes were pretreated with MG-132 for 0 or 60 minutes, with or without rhTpo. Megakaryocyte c-Mpl ubiquitination was analyzed as described in panel A and the blot shown is representative of 2 independent experiments. (C) BaF-Mpl cells were pretreated with vehicle control (DMSO) or the JAK2 inhibitor JAKI for 30 minutes before Tpo stimulation (50 ng/mL, 60 minutes) and c-Mpl ubiquitination was analyzed. The effectiveness of JAKI to inhibit JAK2 activity was determined by analyzing levels of phosphor (p)–STAT5. Data are representative of 2 independent experiments.