Inhibition of TRAF6 and NF-κB signaling depends on the TIFAB C-terminal domain and is independent of the FHA domain. (A) Overview of WT TIFAB and various TIFAB deletion mutants. Summary of NF-κB activation in HEK293 cells after expression of TIFAB mutants under basal (B) and TRAF6-induced conditions (C). Summary of TRAF6 protein expression in HEK293 cells after expression of TIFAB mutants (D). FHA, fork-head associated domain; FLAG, FLAG motif cloned at the N-terminal region of TIFAB mutants; nd, not determined. −, repression; +, normal levels; +/−, moderate repression. (B) NF-κB activation was measured by a κB-site containing reporter assays in HEK293 cells transfected with empty vector, TIFAB, or the indicated TIFAB deletion mutants. Values are normalized to Renilla-luciferase and empty vector (1.0). *, P < 0.05; Student’s t test. (C) NF-κB activation was measured by κB-site containing reporter assays in HEK293 cells transfected with empty vector, TRAF6, and TIFAB or the indicated TIFAB deletion mutants. Values are normalized to Renilla-luciferase and empty vector (1.0). *, P < 0.05; **, P < 0.01; ***, P < 0.001; Student’s t test. (D) Immunoblot analysis of HEK293 transfected with empty vector, TRAF6, and TIFAB or TIFAB deletion mutants. Error bars are mean ± SEM values.