C-Mpl response is reduced by Ott1 loss, and Mpl-TR expression impairs HSC engraftment. (A) Phospho-Stat5 levels in HSCs post-Thpo stimulation. E14.5 fetal liver from Ott1^flox/null Sox2-cre (Ott1 KO) or WT Ott1 controls at baseline (white) or 5 minutes poststimulation with 0 to 50 ng of Thpo (gray) and analyzed through flow cytometry using anti-phospho-Stat5 and surface markers for LT-HSC (LSK CD34^–) (0, 50 ng/mL n = 6 Ott1 KO; n = 7 WT; 1, 10 ng/mL n = 3; error bars ± SD). Representative histograms of flow data at right showing baseline (white) and post 50 ng/mL Thpo stimulation (gray). (B) Phospho-Stat5 in LSKCD34^– GFP⁺ HSCs pre- and post-Thpo stimulation. Median fluorescence intensity of phospho-Stat5 staining in Ott1 KO (Ott1^flox/nullSox2-cre) fetal liver cells expressing either MSCV-IRES-GFP (GFP Ctl) or MSCV-Mpl-FL-IRES-GFP (Mpl-FL) retroviruses at baseline (white) or stimulated with 50 ng/mL Thpo for 5 minutes (gray). Representative histograms (left) and graph of median fluorescence intensity for experiments (n = 3, right). (C) Expression of Mpl constructs. Western blots of 293T cell lysates transfected with no vector, MSCV-HA-Mpl-FL-IRES-GFP, and MSCV-Mpl-TR-IRES-GFP using anti-Mpl antibody or anti-Gapdh antibody. (D) Engraftment of bone marrow overexpressing c-Mpl isoforms. Wild-type bone marrow infected with control GFP (n = 11), Mpl-FL-IRES- GFP (n = 12), and Mpl-TR-IRES-GFP (n = 8) expressing retroviruses was transplanted into irradiated recipients. Serial peripheral blood sampling underwent flow cytometric evaluation for GFP⁺/Mac1⁺/Gr1⁺ percentage of graft normalized to initial GFP⁺ input (bar = mean).