E2F1 binding is localized to 2 consensus binding sites on the Trib2 promoter. (A) (Upper) Schematic presentation of full-length 2.6-kb and deletion mutant Trib2 promoter constructs with the indicated E2F1 binding sites. 3T3 cells were (lower left) transfected with Trib2 luciferase reporter plasmids (pGL3 control, 2.6-kb, 500-bp, 927-bp, or 963-bp promoter region) and (lower right) cotransfected with E2F1 or pcDNA empty expression vector, and luciferase activity was measured. (B) (Upper) Schematic presentation of full-length 2.6 kb with site-directed mutations of the 3 putative E2F1 binding sites. 3T3 cells were (lower left) transfected with Trib2 luciferase reporter plasmids (pGL3 control, 2.6-kb, MutA, MutB, MutC, or double mutant DM) and (lower right) cotransfected with E2F1 or pcDNA empty expression vector, and luciferase activity was measured. Data presented are mean ± SD of duplicate cultures and representative of 3 independent experiments. *P < .05, **P < .005, and ***P < .001. (C) Nuclear extracts prepared from K562 cells transfected with pcDNA3 empty or E2F1 were assayed for E2F1 binding for sites A, B, and C by EMSA. Arrow indicates E2F1 binding. FP, free probe; comp, cold competition; MT, mutant type. (D) 3T3 cells were cotransfected with E2F1 WT or E2F1 E132 DNA binding deficient mutant or empty expression vector and Trib2 luciferase reporter constructs (pGL3 control, 2.6-kb, 500-bp, or DM promoter region), and luciferase activity was measured. Data presented are mean ± SD of duplicate cultures and representative of 2 independent experiments. **P < .005 and ***P < .001 using an unpaired Student t test. (E) K562 cells were cotransfected with E2F1 WT or E2F1 E132 DNA binding deficient mutant or empty expression vector and Trib2 luciferase reporter constructs (pGL3 control, 2.6-kb, 500-bp, or DM promoter region), and luciferase activity was measured. Data presented are mean ± SD and representative of 2 independent experiments. ***P < .001 using an unpaired Student t test.