Figure 3
Figure 3. Both the Trib2 KD- and COP1-binding sites are required for Trib2 activity. (A) Schematic of Trib2 point mutants. Left, amino acid sequences show original sequence on top and mutated sequence on bottom. (B) Effect of Trib2 mutants on 32D cell differentiation. 32D cells transduced with the vector control (MigR1-myc; MM), WT Trib2 (FL) or the Trib2 mutants were plated in equal numbers (1 × 105; day 0, 48 hours posttransduction) in IL-3 or G-CSF. Transduced cells were identified by the expression of the GFP surrogate marker. CD11b expression was assessed after 2 and 5 days. Data are representative of 3 independent experiments. (C) Effect of Trib2 mutants on C/EBP-α expression. 32D cells were transduced with the indicated retroviral constructs, sorted for GFP 48 hours after transduction, and assessed for C/EBP-α, myc-tagged FL Trib2, and myc-tagged Trib2 mutant (VPM, KDM, and K177R) protein expression by Western blot, 3 days after sorting. MigR1-Myc is the empty vector (MM). β-actin is the protein loading control. (D) Effect of Trib2 mutants on 32D cell cycle. 32D cells were transduced with the vector control (MigR1-myc; MM), WT Trib2 (FL), or the Trib2 mutants, sorted for GFP 48 hours later, and 1 × 105 GFP+ cells were plated in 5 ng/mL IL-3 or 25 ng/mL G-CSF 3 days after sorting. Cell cycle was assessed by propidium iodide staining on days 2 and 5. (E) Effect of Trib2 mutants on serial replating by transduced BM. 5-FU–treated BM cells transduced with the MigR1 retroviral vector, WT Trib2, or the indicated Trib2 mutants were plated in equal number (25 000 unsorted cells in triplicate) in methylcellulose (M3231), containing IL-3, IL-6, SCF, and GM-CSF. Colonies with > 50 cells were scored and assessed over 3 rounds of serial replating. 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. (F) Interaction of Trib2 mutants and C/EBP-α. GST pull-down of Trib2 constructs incubated with IVT 35S C/EBP-α. Top panel: autoradiograph of 35S C/EBP-α. Bottom panel shows Coomassie gel of GST proteins.

Both the Trib2 KD- and COP1-binding sites are required for Trib2 activity. (A) Schematic of Trib2 point mutants. Left, amino acid sequences show original sequence on top and mutated sequence on bottom. (B) Effect of Trib2 mutants on 32D cell differentiation. 32D cells transduced with the vector control (MigR1-myc; MM), WT Trib2 (FL) or the Trib2 mutants were plated in equal numbers (1 × 105; day 0, 48 hours posttransduction) in IL-3 or G-CSF. Transduced cells were identified by the expression of the GFP surrogate marker. CD11b expression was assessed after 2 and 5 days. Data are representative of 3 independent experiments. (C) Effect of Trib2 mutants on C/EBP-α expression. 32D cells were transduced with the indicated retroviral constructs, sorted for GFP 48 hours after transduction, and assessed for C/EBP-α, myc-tagged FL Trib2, and myc-tagged Trib2 mutant (VPM, KDM, and K177R) protein expression by Western blot, 3 days after sorting. MigR1-Myc is the empty vector (MM). β-actin is the protein loading control. (D) Effect of Trib2 mutants on 32D cell cycle. 32D cells were transduced with the vector control (MigR1-myc; MM), WT Trib2 (FL), or the Trib2 mutants, sorted for GFP 48 hours later, and 1 × 105 GFP+ cells were plated in 5 ng/mL IL-3 or 25 ng/mL G-CSF 3 days after sorting. Cell cycle was assessed by propidium iodide staining on days 2 and 5. (E) Effect of Trib2 mutants on serial replating by transduced BM. 5-FU–treated BM cells transduced with the MigR1 retroviral vector, WT Trib2, or the indicated Trib2 mutants were plated in equal number (25 000 unsorted cells in triplicate) in methylcellulose (M3231), containing IL-3, IL-6, SCF, and GM-CSF. Colonies with > 50 cells were scored and assessed over 3 rounds of serial replating. 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. (F) Interaction of Trib2 mutants and C/EBP-α. GST pull-down of Trib2 constructs incubated with IVT 35S C/EBP-α. Top panel: autoradiograph of 35S C/EBP-α. Bottom panel shows Coomassie gel of GST proteins.

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