Reduced RasGRP1-expressing cells in Gfi1-null mice. (A) Levels of RasGRP1 mRNA were measured by quantitative real-time PCR in thymus, spleen, and bone marrow extracted from individual Gf1+/+, Gfi1+/−, and Gfi1−/− mice. The results are representative of at least 5 mice/group. (B) RasGRP1 was immunoprecipitated from lysates of thymus (300 μg, top; 800 μg, bottom), spleen (300 μg), and bone marrow MNCs (300 μg, top; 3 mg bottom) from individual Gfi1+/+ and Gfi1−/− mice. The immunoprecipitates were immunoblotted with RasGRP1-specific antibody. The bottom panel relates to tissues from a Gfi1+/+ mouse (representative of 3 experiments). (C) RasGRP1 mRNA levels measured by quantitative real-time PCR in individual bone marrows from Gfi1+/+ (n = 8), Gfi1+/− (n = 11), and Gfi1−/− (n = 9) mice. Results from individual mice, ■; and group means, 
. (D) Flow cytometric detection of RasGRP1 in a population of bone marrow MNCs from a Gfi1+/− but not Gfi1−/− mouse. After fixation and permeabilization, the cells were immunostained with a mouse monoclonal antibody to RasGRP1 or isotype control antibody (mouse IgG1) followed by a goat anti–mouse IgG Alexa Fluor 488–conjugated antibody. The results reflect the percentage of RasGRP1-positive cells and are representative of 5 independent experiments. (E) Forward scattering counter (FSC) and SSC of bone marrow MNCs from a Gfi1+/− mouse after RasGRP1 immunostaining (as described in panel D). Left panel shows total; right panel shows RasGRP1 only. The results are representative of Gfi1+/+ (n = 4) and Gfi1+/− (n = 3) mice. (F) Flow cytometric analysis of Gr1 and RasGRP1 expression within bone marrow MNCs from a Gfi1+/− mouse, showing that RasGRP1-positive cells display low-level surface Gr1. The MNCs were first immunostained for surface Gr1 (PE-labeled) and after fixation/permeabilization immunostained for RasGRP1 (as described in panel D). Representative of 5 independent experiments is shown. The percentage of cells in the box is shown. (G) Flow cytometric detection of the surface markers neutrophils 7/4, CD11b, CD4, CD45R/B220, cKit, G-CSFR, and intracellular RasGRP1in bone marrow MNCs from a Gfi1+/− mouse. G-CSFR was detected by PE-labeled G-CSF cross-linked to the cell surface with Bis(sulfosuccinimidyl)suberate3 . The results are representative of 3 experiments. The percentage of cells in each quadrant is shown. (H) Flow cytometric analysis of RasGRP1-positive cells within LSK (lin−c-Kit+Sca+), MPP (CD150−CD48−CD244+), CMP (lin−Sca−c-Kit+CD34+CD16/32mid), and GMP (lin−Sca−c-Kit+CD34+CD16/32hi) bone marrow cells populations from Gfi1+/+ MNCs. Representative of 3 experiments is shown. The values reflect percentage of cells in each quadrant.
