FIGURE 1.

Analysis of gain-of-function of CBL-Y371H in CBL-null and CBL/CBL-B-null primary mouse HSPCs. A, schematic illustration of various second-site mutants of CBL-Y371H mutants used in this study. B, mouse primary HSPCs were isolated and infected with the indicated retroviruses as described under “Experimental Procedures.” Data shown are representative FACS plot of the GFP+ population from Cbl null and Cbl/Cbl-b DKO HSPCs. C–H, lineage-negative HSPCs were purified by immuno-depletion of lineage-positive mature cells from bone marrow mononuclear cells collected from CBL-null (C–E) or CBL/CBL-B-null (DKO) mice (F–H). The cells were retrovirally infected to express the indicated constructs carrying an IRES-GFP, and infected (GFP+) cells obtained by FACS sorting were assessed in three replicates for cytokine-independent (C and F), SCF-stimulated (D and G), or TPO-stimulated (E and H) proliferation as described under “Experimental Procedures.” For each in C–H, left is one representative experiment with six replicates (mean ± S.D.); right is pooled data of three independent experiments shown as percentage of uninfected control (mean ± S.E.). *, p < 0.05.