Fig 1.

Enhancement of TIFA phosphorylation by TNF-α stimulation. (A) Schematic overview of TIFA domain structure (top) and N-terminal sequences of TIFA orthologues from different species (bottom). T9 is highlighted in dark gray. (B) MS/MS spectrum of the peptide containing pT9 in TIFA. The precursor ion 827.2816²⁺ is from ADPMTSFEDAD(pT)EE. (C) NanoPro immunoassay demonstrates the phosphorylation status of overexpressed Myc-tagged WT and T9A mutant TIFA with or without TNF-α and phosphatase treatment. The peaks at pI 4.75 and pI 4.63 are assigned to unphosphorylated TIFA and singly phosphorylated TIFA, respectively. The bar graph in the lower panel represents the ratios of peak areas of singly phosphorylated TIFA to total TIFA. The results represent means ± standard deviations (SD) from at least 3 independent experiments. (D) NanoPro immunoassay of endogenous TIFA. The conditions were the same as those for panel C. The pI values differ from the peaks in panel C due to lack of the Myc tag. (E) Recombinant TIFA or the T9A mutant was incubated with [γ-³²P]ATP and lysates from cells treated with or without TNF-α for 30 min. During incubation, alkaline phosphatase (PPtase) was added as indicated. The reaction mixtures were separated by SDS-PAGE, and the bands were revealed by autoradiography. (F) The experimental conditions were the same as those for panel E except for the inclusion of caffeine or inhibitor cocktails for AKT, IRAK, TAK, or PKC (Go 6976 and Go 6983).