Figure 5. Trib2 reduces normal C/EBPα levels and inhibits its activity.

(A) Sorted GFP⁺ 32D and U937 cells transduced with either MigR1 or Trib2 were assessed for C/EBPα protein expression by western blot. C/EBPαp42 is the full-length protein and C/EBPαp30 is the truncated protein. Actin is the protein loading control.

(B) Analysis of C/EBPαp42 and p30 protein expression in primary leukemic samples from BM (93% GFP⁺), spleen (63% GFP⁺) and lymph node (88% GFP⁺) compared to normal levels expressed in CMPs and GMPs from B6 BM (left panel). Levels of C/EBPαp42 and p30 proteins were also compared in unfractionated normal B6 mouse BM cells and primary (94% GFP⁺) and secondary (98% GFP⁺) leukemic BM samples (right panel).

(C) Schematic of the IL-12 promoter containing the C/EBPα binding site.

(D) RAW264.7 macrophages were co-transfected with i) the IL-12 promoter firefly luciferase constructs containing the C/EBP WT or mutant binding sites, ii) either empty vector, Trib2, C/EBPα or both Trib2 and C/EBPα, and iii) a pRL-TK Renilla luciferase internal control plasmid. Luciferase activity was measured following LPS (100 ng/ml) treatment for 8 hrs, 24 hrs post-transfection. Reporter luciferase activity for each sample was normalized to the Renilla luciferase activity of the same sample. Data presented are mean +/− SD of triplicate cultures.

(E) C/EBPα DNA binding activity was assessed by EMSA using a double-stranded oligonucleotide probe containing the C/EBP binding site from the human G-CSF receptor. Nuclear extracts from sorted GFP⁺ U937 cells transduced with MigR1 (lanes 1, 2), Trib2 (lanes 3, 4), or C/EBPα (lanes 5, 6), were incubated with ³²P-labeled probe. In lanes 2, 4, and 6, 2 μL of C/EBPα antibody was added. SS indicates the supershifted C/EBPα complex. The extracts used in lanes 1, 3, and 5 were tested with an OCT-1 probe in a second EMSA assay as a control for the integrity and quantity of nuclear binding proteins.