Figure 9: DDX3 disrupts the formation of SHP/HNF4 heterodimer and promotes the formation of the active HNF4 homodimer.

(a and b) HuH7 cells were transfected with HA-SHP expression plasmid alone or together with Flag-HNF4 expression plasmid as indicated. (a) Immunoblotting was performed with antibodies against HA and Flag. (b) The total cell lysates (2 mg) of co-expressed Flag-HNF4 and HA-SHP were incubated with anti-Flag M2 agarose resins. After extensive washing, increasing amounts of purified GST-DDX3 proteins (as indicated on the top, lane 3–5) were added to the mixtures of the beads with bound fractions. The immunoprecipitated proteins were analyzed by immunoblotting with antibodies against Flag, HA and DDX3. The GST-DDX3 alone (lane 1) and cell lysates of expressed HA-tagged SHP (lane 2) incubated with anti-Flag M2 agarose resins were used as negative control. (c,d and e) Similar experiments were performed as shown in panel a and b, except HuH7 cells were transfected with HA-HNF4 expression plasmid alone or together with Flag-SHP expression plasmid (c and d), or purified GST protein was used instead of GST-DDX3 in (e). (f) Overexpression of DDX3 increases HNF4 homodimer formation. HuH7 cells were transfected with either GFP or GFP-DDX3 expressing plasmids together with HA and Flag vectors (−) or expressing plasmids for HA-HNF4 and Flag-HNF4 (+). Forty-eight hours later, cells were fixed and analyzed by PLA with anti-HA (Abcam) and anti-Flag antibodies. Nuclei were stained with DAPI (blue). In cells cotransfected with HA-HNF4 and Flag-HNF4, the numbers of PLA signals per cell were quantified using MetaMorph software and are shown as means ± S.D. relative to that of GFP-cotransfected cells. n = 50. ***p < 0.001.