The Trib2 N-terminus is dispensable for WT Trib2 activity. (A) Effect of Trib2 mutants on 32D cell differentiation. 32D cells were transduced with the retroviral vector (MigR1), WT Trib2 (FL), or Trib2 mutants and plated in equal numbers (1 × 10⁵; day 0, 48 hours posttransduction) in IL-3 or G-CSF for 5 days. Transduced cells were identified by the expression of the GFP surrogate marker. CD11b expression was assessed after 5 days in culture. Data are representative of 3 independent experiments. (B) Effect of Trib2 mutants on C/EBP-α expression. Sorted GFP⁺ 32D cells transduced with the indicated retroviral constructs that had been cultured in IL-3 for 2 days were assessed for C/EBP-α protein expression by Western blot. β-actin is the protein loading control. (C) Serial replating ability of Trib2-transduced BM cells. 5-FU–treated BM cells transduced with the retroviral vector (MigR1) or Trib2 constructs were plated in equal number (25 000 unsorted cells in triplicate) in methylcellulose (M3231) containing IL-3, IL-6, SCF, and GM-CSF. The mean numbers of colonies normalized for GFP percentage (as a marker for transduced cells) ± SEM are shown. Data are representative of triplicate cultures from 2 independent experiments. (D) Serial replating ability of Trib2-transduced BM cells from chimeric mice. Then, 25 000 sorted GFP⁺ BM cells obtained from chimeric mice (MigR1, Trib2, dN, dC, and KD) 6 weeks after BMT and 25 000 control B6 total BM cells were plated in triplicate in methylcellulose (M3434, which contains SCF, IL-3, IL-6, and erythropoietin). The mean numbers of colonies ± SEM are shown. Data are representative of triplicate cultures from 2 independent experiments.