Pak2 deletion stimulates megakaryopoiesis in vivo. (A) WB to detect Pak2 levels in bone marrow (top) and megakaryocytes (bottom) 14 DPI. Actin serves as a loading control for relative protein levels. Megakaryocytes lysates collected from cultured bone marrow of pIpC-injected Mx1-cre⁺;Pak2^fl/fl and wild-type (notated as (Pak2^−/− and Pak2^+/+, respectively) mice at 14 DPI. (B) Representative bone marrow histology from Pak2^+/+ and Pak2^−/− mice (n = 5 mice/genotype), both treated with plpC and analyzed 14 DPI. White arrows indicate megakaryocytes. Scale bar = 20 μm (×40 original magnification). (C) Measurement of megakaryocyte diameter in the bone marrow (n = 5 mice/genotype). (D) Percentage of bone marrow expressing CD41 measured by flow cytometry. (E) CD34+ bone marrow stem cells sorted by flow cytometry and cultured with 100 ng/mL TPO for 5 days. Representative WB of phospho-Pak1/2/3 (Serine141) and Pak2 expression during a 5-day time course. Actin serves as a loading control for total protein levels. CD41 expression marks mature megakaryocytes. (F) Fluorescence microscopy of megakaryocyte (top), proplatelet (middle), and platelet (bottom) stained for β1-tubulin, Pak2, and nuclei (Hoechst). Scale bar = 10 μm. All values are mean ± standard error of the mean (SEM) for at least 5 mice/genotype. *P < .05.