Fig. 3.
TBK1 autoactivation via S172 phosphorylation. FLWT, KUWT, and KDWT were analyzed by (A) anti-pS172 immunoblotting and (B) LC-MS before and after incubation with ATP. Extracted ion chromatograms for S172 and pS172 peptides are shown for TBK1 FLWT, with quantitation of all samples summarized (mean ± SD error bars, n = 3) (Inset). (C) Transautoactivation of TBK1 was probed using combinations of wild type and kinase-dead (D135N) variants and anti-pS172 Western blots. (D) A time course of KDD135N phosphorylation by preactivated (pre-phos) or dephosphorylated (de-phos) KUWT was evaluated by pS172 immunoblotting. The KUS172A-mediated transphosphorylation of KUD135N is observable at longer time points.