Extended Data Fig. 8: Mass spectrometric analyses of the AMPylation of threonine-phosphorylated and serine-phosphorylated peptides by LnaB.

a, MS/MS analysis of the 10-amino acid threonine-phosphorylated peptide APKpTPPSSGE of Tau, which was used as the substrate of LnaB. b, MS detection of the 10-amino acid threonine-phosphorylated peptide of Tau and the 10-amino acid ADP-T peptide after modification by LnaB. Extracted ion chromatograms of the protonated peptides are shown with peak intensities, which indicate the relative amounts of the pT-containing (m/z = 525.73) and the ADP-T (m/z = 690.26) peptides. c, MS/MS analysis of AMPylation on the 10-amino acid threonine-phosphorylated peptide of Tau by LnaB. d, MS/MS analysis of the 8-amino acid serine-phosphorylated peptide NNKGpSAAW of TAK1, which was used as the substrate of LnaB in Fig. 5f. e, MS detection of the 8-amino acid serine-phosphorylated peptide of TAK1 and the 8-amino acid ADP-S peptide after modification by LnaB. f, AMPylation of the okadaic acid- stimulated 293T cell lysates by FLAG-tagged LHEI proteins immunoprecipitated from the LHEI-overexpressed cells. The samples were analysed by SDS–PAGE and immunoblotting. *, nonspecific bands. g, Screening of AMPylation of various kinases by LnaB during infection. 293T cells expressing NPM-ALK, GFP-TBK1, 3×FLAG-IKK or 3×FLAG-AKT1 were infected with the wild-type Lp02 or ΔlnaB strain of L. pneumophila for 6 h. After immunoprecipitation, AMPylation was analysed by immunoblotting. h, MS/MS analysis of the activation loop peptide LIKDDEpYNPCQGSK of Fgr during infection of the L. pneumophila ΔlnaB strain.