Figure 5

Binding of Hnf-1 to the −84/−68 region of the Angptl8 gene. (a) Electrophoretic mobility-shift assays (EMSA) showed that Hnf-1 in whole cell extracts from Hepa1–6 cells significantly bound to the wildtype probe of the Angptl8 promoter lesion –94/−60 that included the Hnf-1 binding consensus motif. This complex was supershifted with a specific antibody against Hnf-1. By contrast, no protein binding was observed using mutant probes (M1 or M2) that contain nucleotide mutations in the Hnf-1 binding consensus motif. (b) The amount of Hnf-1 bound to the region –94/−60 was reduced in a dose-dependent manner by adding 20- or 40-fold molar excess cold (non-³²P labelled) probes. Adding non-labelled probes including mutated nucleotides in the Hnf-1 binding consensus motif did not alter the amount of Hnf-1 binding. W.C.E indicates the whole cell extract and N.S indicates the non-specific binding.