TIFA promotes DNA damage–induced NEMO ubiquitination.
a, FLAG-NEMO was co-transfected with control vector or EGFP-TIFA to HeLa cells. Cellular extracts were immunopurified with FLAG M2 resin and eluted with FLAG peptide. Eluted proteins were resolved by SDS-PAGE and silver stained. The bands were retrieved and analyzed by MS. b, HeLa cells were co-transfected with FLAG-NEMO, HA-Ub, and EGFP-TIFA as indicated. All the cells were treated with ETO before cellular extracts were prepared. The immunoprecipitation was performed with FLAG antibody and the immunoprecipitated (IP) proteins were examined with the indicated antibody. c, HeLa cells were transfected with plasmids expressing WT TIFA or its T9A mutant (TIFA T9A) together with the other indicated constructions. The immunoprecipitation assay was performed as in b. IgG heavy chain is indicated with *. d, HeLa cells were transfected with plasmids expressing WT TIFA or its FHA domain-deleted mutant (TIFAΔFHA) together with other indicated constructions. The immunoprecipitation assay was performed as in b. IgG heavy chain is indicated with *. e, HeLa cells were transfected with plasmids expressing WT TIFA or its T9A mutant (TIFA–T9A) together with other indicated constructions. The cells were harvested and then plasma and nucleus was isolated. The immunoprecipitation (IP) assay was performed.