Because UXT interacted specifically inside the nucleus with p65 in a signal-dependent manner, we went on to address whether this interaction was important for regulating NF-κB activity and genes responsive to it. We transfected 293T cells with UXT or other control plasmids, prepared nuclear extracts, and performed electrophoretic mobility shift assay (EMSA) as indicated. Consistently, there was no NF-κB binding to its cognate probe without TNF-α treatment. Notably, the overexpression of UXT alone did not induce any detectable basal NF-κB binding activity.
Overexpression of UXT does not markedly affect NF-κB activation induced by TNF-α. (A) 293T cells were transfected with an equal amount of UXT, A20, or control vector. 24 h after transfection, cells were stimulated with 10 ng/ml TNF-α for the indicated times. Equal amounts (10 μg) of nuclear extracts were subjected to EMSA. For competition analysis, 100-fold excess of unlabeled wild-type or mutant κB probes were added to the reaction mixtures. For supershift assays, nuclear extracts were incubated with antibody as indicated.
EMSA was performed to test endogenous NF-κB or sp1 binding to their cognate probes.
Via EMSA, we then analyzed endogenous NF-κB DNA binding activity in cells with reduced UXT expression. Expectedly, TNF-α alone induced endogenous NF-κB to bind to its cognate probe strongly and specifically. Considerably, this interaction was markedly diminished in nuclear extracts from UXT-specific knockdown cells. In addition, this reduction was correlated with the effectiveness of the siRNA administered (Fig. 5 B).
EMSA was performed to test endogenous NF-κB or sp1 binding to their cognate probes.
Furthermore, EMSA confirmed that the loss of UXT also suppressed the constitutive DNA binding activity of NF-κB but not the control sp1 (Fig. 9 E). These results indicate that the elevated expression of UXT strongly correlates with constitutive NF-κB activity in prostate cancer cell lines and again substantiates the notion that UXT is essential for NF-κB function in the nucleus.