GdX blocks the interaction of TC45 with p65. (A) GdX specific interacted with TC45. Myc-GdX was co-expressed with Flag-tagged IKKα, IKKβ, IKKε, WIP1, PP4 or HA-tagged PP1, PP2A and TC45 in HEK293T cells. Immunoprecipitation (IP) experiments were performed using an anti-Flag or HA antibody. (B) HA-TC45 interacted with Flag-p65. HEK293T cells were transfected for IP with an anti-Flag antibody. (C) The association of endogenous TC45 and p65 was detected in immune cell lines. Lysates from DC2.4 or RAW264.7 cells were subjected to IP experiments with an antibody against TC45. (D) Flag-p65 interacted with GST-TC45 purified from E. coli. (E) The interaction of Flag-p65 and HA-TC45 was increased under TNF-α stimulation (10 ng/mL) for 15 min. (F) The interaction of endogenous TC45 and p65 was increased under LPS or TNF-α stimulation. IP experiments were performed by an anti-TC45 antibody. (G) Over-expression of GdX disrupted the association of TC45 and p65. HEK293T cells were used for IP experiments after transfected with HA-TC45 in the presence or absence of Myc-GdX. (H) The association of endogenous TC45 and p65 was increased in GdX-deficient (KO) splenocytes comparing with that in the wildtype (WT) cells. (I) GdX(L29P) mutant failed to block the interaction of p65 and TC45. (J-K) A molecular docking analysis shows the interaction surface of TC45 with the N-terminus of p65 (J) and GdX (K). F183 in TC45 maintains a core residue for the interaction with p65 and GdX although GdX shifts slightly to one side in the interaction (K).