Figure 2.

Analysis of preleukemic Dok-1 ^−/−/Dok-2 ^−/− mutants. (A) In vitro colony-forming assay performed with BM cells isolated from a WT (white bar) and a Dok-1 ^−/−/Dok-2 ^−/− mutant (DKO; black bar) at 4 mo of age. P-value is also shown. (B) [³H]thymidine incorporation analysis of proliferative response of cells collected from in vitro colony assay of BM cells from 2-mo-old WT, Dok-1 ^−/−, Dok-2 ^−/−, and DKO mice. (C) Proliferative response determined by [³H]thymidine incorporation of Mac-1⁺ BM cells upon IL-3, GM-CSF, and SCF stimulation. (D) Apoptotic response to growth factor deprivation in BM cells. Cell death was analyzed by annexin V staining of WT (white bar) and Dok-1 ^−/−/Dok-2 ^−/− (DKO; black bar) Mac-1⁺ BM cells. (E) GM-CSF–induced Ras activation of Mac-1⁺ BM cells isolated from 4-mo-old WT, Dok-1 ^−/−, Dok-2 ^−/−, and DKO mice. A representative Western blot from three experiments is shown. Very low amounts of Ras-GTP were detected in serum-starved cells before GM-CSF treatment. (F) Relative Ras activity in the above cells treated with GM-CSF is quantified by the ratio of GTP-bound Ras over total Ras. The value of WT BM cells was set as 1 and the ratios of other types of BM cells were calculated correspondingly. (G) Levels of ERK 1/2 phosphorylation upon (lanes 3 and 4) or in the absence (lanes 1 and 2) of GM-CSF stimulation in BM cells from WT (lanes 1 and 3) and Dok-1 ^−/−/Dok-2 ^−/− (lanes 2 and 4) mice.