The Rel homology domain of p65 specifically binds to DDX3, and the ATP-dependent RNA helicase activity of DDX3 is important for its inhibitory effect on NF-κB target gene expression.

<p>(A) Co-immunoprecipitation of HA-tagged p65 wt and deletion mutants. Lysates of 293T cells transfected with Flag-DDX3 (4 μg) and HA-p65 wt or deletion mutants (1–332 or 299–551) (4 μg) were used for immunoprecipitation analysis. Anti-HA antibodies were used to capture the immunocomplex. DDX3 in the immunoprecipitates was detected by immunoblotting with mouse monoclonal anti-Flag antibodies. Anti-Flag and anti-HA antibodies were used to detect Flag-DDX3 and HA-p65 wt and deletion mutants (1–332 or 299–551), respectively, in whole cell lysates (middle and lower layers). (B) Truncated DDX3 (1–310) and DDX3 (310–662) expression levels were detected by Western blotting. (C to F) Effect of truncated DDX3 on NF-κB-mediated gene expression. The 5xNF-κB-luc reporter gene was activated by TNFα (20 ng/ml) or 100 ng of IKKβ, NIK or p65. The cells were co-transfected with 400 ng of DDX3, DDX3 (1–310), DDX3 (310–662) or empty vector (pcDNA3.0, negative control). Firefly and <i>Renilla</i> luciferase activity was measured (<i>n</i> = 3, mean ± SD) at 24 hours post transfection. (G) The expression level of DDX3-K230E was detected by Western blotting. (H to K) Effect of the ATP-dependent RNA helicase mutant of DDX3 on NF-κB-mediated gene expression. The 5xNF-κB-luc reporter gene was activated by TNFα (20 ng/ml) or 100 ng of IKKβ, NIK or p65. The cells were co-transfected with 400 ng of wild-type or mutant DDX3 expression plasmid (HA-DDX3-K230E) or empty vector (pcDNA3.0, negative control). Firefly and <i>Renilla</i> luciferase activity was measured (<i>n</i> = 3, mean ± SD) at 24 hours post transfection. ** p < 0.01, *** p < 0.001 and **** p < 0.0001 (unpaired Student’s t-test). The mock (non-induced) control was transfected with 100 ng of empty vector corresponding to each inducing factor.