Raf-1 and in vitro megakaryocytopoiesis. (A) The fold expansion of raf-1^-/- and raf-1^+/+ CD41⁺ low-density bone marrow mononuclear cells grown in serum-free liquid culture supplemented with 50 ng/mL TPO is presented in semilog format. Data represent the mean ± SEM from 3 independent experiments. (B) Megakaryocytes generated by 4 days of culture in TPO-containing media were washed and resuspended in media with (+TPO) or without (-TPO) TPO, and TUNEL assays were performed following an additional 0, 24, or 48 hours of incubation. The percentage of TUNEL-positive bone marrow–derived megakaryocytes following culture for the indicated times in media with or without TPO is presented (left). Data are the mean ± SEM of 5 independent experiments. The same experiments performed on megakaryocytes derived from E13.5 to E15.5 fetal liver cells (n = 3) produced similar results (right). (C) DNA ploidy analysis of raf-1^+/+ and raf-1^-/- megakaryocytes stained with fluorescein isothiocyanate–conjugated antimouse CD41 antibody and propidium iodide. Data are from CD41⁺ megakaryocytes and are representative of 3 independent experiments that each produced the same results. DNA ploidy values ranging from 2N to 128N are indicated above their respective peaks. (D) Western blots demonstrate Raf-1, A-Raf, and B-Raf protein expression in purified mature megakaryocytes derived from raf-1^+/+ (+/+) or raf-1^-/- (-/-) bone marrow. Total protein (20 μg) was loaded in each lane, separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and probed with antibodies against Raf-1, B-Raf and A-Raf, and β-tubulin, the latter serving as an independent measure of protein loading. (E) A representative Western blot demonstrates similar kinetics and extent of TPO-induced ERK phosphorylation in purified raf-1^-/- and raf-1^+/+ BM-derived megakaryocytes. The proteins were separated by 10% SDS-PAGE, and blots were probed as indicated using anti–phospho ERK, anti–total ERK, or anti–Raf-1 antibody.