Figure 5.

Hematological parameters of bone marrow transplanted mice. (A) Experimental design. FVB/N mice (45.1) were transplanted intrafemorally with lentivirally transduced FVB/N-45.2 bone marrow, infected either with an empty vector, or with a vector expressing WT NF-E2 or the indicated NF-E2 mutants (n = 4–11 mice/construct, as indicated). 12 wk after transplantation, engraftment exceeded 90% in all cases. (B) Total cell extracts of murine bone marrow transduced with the indicated NF-E2 constructs was interrogated with an antibody that recognizes both human and murine NF-E2. Equal loading was assured by reprobing with an antibody against β-actin. A representative blot of n = 3 independent experiments is shown. (C) Total cell extracts of murine bone marrow transduced with the NF-E2-262aa mutant (left lane), as well as protein extracts of a patient carrying the NF-E2-262aa truncation mutations (UPN 202) were interrogated for NF-E2 (top) and β-actin (bottom) expression by Western blotting. The outer right lane serves as a positive control and depicts lentivirally transduced CB3 cells expressing both WT and the 262aa truncated NF-E2. A filled arrowhead demarks full-length WT NF-E2, an open arrowhead points to the smaller, truncated NF-E2 proteins, and an asterisk demarks a nonspecific band. A representative blot of two independent experiments is shown. (D–G) Peripheral blood was obtained by retro-orbital puncture and analyzed on an Advia 120 system at the indicated time points (n = 4–11 mice per genotype). Mean and SEM are shown. (D) Platelet counts. (E) Hematocrit. (F) RBC count. (G) ANC. Statistical analysis was conducted using Student’s t tests., *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus WT.