Fig 2.

pT9 and the FHA domain are required for TIFA self-association. (A) Flag-tagged WT and Myc-tagged WT and T9A TIFA were coexpressed in HEK 293T cells. Anti-Flag-coated Dynabeads was used in immunoprecipitation (IP). The coprecipitated proteins were detected by anti-Myc antibody in immunoblotting. The whole-cell extracts (WCE) used as IP inputs are shown in the right panel. PPtase or EDTA was included as indicated. (B) Sequence alignment of the conserved pT-contacting residues within the FHA domains of different FHA domain-containing proteins. Conserved residues among homologues are in light gray. The residues chosen for mutation in this work are in dark gray. RAD53*, RAD53 FHA1. (C) Effect of FHA domain mutations on the self-association of TIFA. The experiments were performed as described for panel A except that the coprecipitated TIFA and its mutants were detected by anti-Gal antibody. RKN, R51A K88A N89A mutant. (D) ITC analysis of the in vitro binding of WT TIFA and its R51A and R51A K88A (RK) mutants with the T9-containing peptide (¹MTSFEDADTEETVT¹⁴) or pT9-containing peptide [¹MTSFEDAD(pT)EETVT¹⁴]. Binding was observed only when the WT TIFA was incubated with the pT9-containing peptide.