Fig. 2

HBP1 represses the AFP gene through binding an affinity site in the AFP promoter. a Relative activity of HBP1 on the AFP promoters with various lengths. b HBP1 suppresses AFP promoter activity in a dose-dependent manner. Luciferase activity was detected in 293 T cells co-transfected with the indicated reporter genes and HBP1 plasmids. c The integrity of affinity site is indispensable for HBP1 suppressing AFP promoter in vivo. Shown is schematic diagram of the wild type AFP promoter and its mutant promoter (left panel) and the relative activities of HBP1 on the wild type AFP promoter and mutant AFP promoters (right panel). d Relative activities of HBP1 and associated mutants on the AFP promoter in co-transfected 293 T cells. Shown is schematic diagram of wild-type HBP1 and associated mutants (left panel). Luciferase activity was determined after transfection (right panel). e Expression of exogenous HBP1 decreases AFP protein level. Two hundred-ninety three T cells were transfected with HBP1 and associated mutants. The protein level was measured by Western blotting. f HBP1 occupies its affinity site in the AFP promoter. EMSA assays were performed by using a biotin-labeled double-stranded probe consisting of one HBP1 affinity site. Nuclear extracts from 293 T cells transfected with or without HBP1, pmHMG, or DelEx7 expression plasmid were used. Cold competitors were included in the indicated lanes at 200-fold excess. The presence of specific complexes, including supershifted HA or HBP1, was indicated. g HBP1 binding to the endogenous AFP promoter requires the HMG domain. ChIP assays were used to test the binding of exogenous HBP1 to endogenous AFP gene. Two hundred-ninety three T cells were transfected with HA-HBP1, HA-pmHMG or HA-DelEx7. The region from position − 1600 to position − 1448 contains the HBP1 affinity site and was analyzed by specific PCR and Realtime-PCR. Anti-HA antibody was used in the indicated lanes. Error bars represent S.D. *, p < 0.05, **, p < 0.01, ***, p < 0.001