Figure 5: DDX3 enhances HNF4 binding to the MTP promoter. | Scientific Reports

Figure 5: DDX3 enhances HNF4 binding to the MTP promoter.

From: RNA helicase DDX3 maintains lipid homeostasis through upregulation of the microsomal triglyceride transfer protein by interacting with HNF4 and SHP

Figure 5

(a) Western blot analysis of subcellular localization of DDX3. HuH7 cells were transfected with plasmid expressing HA-HNF4 alone or together with HA-DDX3 expression construct as indicated. After 48 hr, cytosolic fractions and nuclear extracts (20 μg of each) were prepared and subjected to SDS-PAGE then immunoblotting with anti-HA antibody. (b) HNF4 DNA binding ability is enhanced by DDX3. The nuclear extracts of transfected HuH7 cells were incubated with annealed biotinylated probe containing HNF4-responsive element and subjected to binding with streptavidin agarose. After extensive washing, the bound fractions were analyzed by SDS-PAGE, followed by immunoblotting with anti-HA antibody. (c) Western blot analysis of the ectopically expressed Flag-HNF4 and HA-DDX3. HuH7 cells were transfected with plasmids expressing HA-DDX3 and Flag-HNF4 as indicated. Western blot analysis was performed 48 hr posttransfection. The expression level of HA-DDX3 and Flag-HNF4 was detected using anti-HA and anti-Flag antibodies, respectively. (d) Overexpression of DDX3 enhances the DNA binding activity of Flag-HNF4 on MTP promoter. Chromatin immunoprecipitation assay was performed by isolation of soluble chromatin fragments prepared from transfected-HuH7 cells and then immunoprecipitated with anti-FLAG M2 agarose resins. Immunoprecipitates were analyzed by quantitative real-time PCR with specific primers for MTP promoter (as described in Materials and Methods). Results are expressed as means ± S.D. for at least three independent experiments and **p < 0.01, is considered to be significant.

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