Figure 1. NleE specifically targets TAB2/3 in the NF-κB pathway.

(A) Back-methylation analysis of NleE modification of cellular TAB2. Flag-TAB2 co-expressed with a vector or NleE in 293T cells was immunopurified and incubated with recombinant NleE in the presence of ³H-SAM. NleEΔ6, NleE mutant with deletion of ₂₀₉IDSYMK₂₁₄. (B–D) Mass spectrometry analysis of NleE modification of TAB2 (B), HOIL-1L (C) and Sharpin (D) during EPEC infection. Shown are the extracted ion chromatograms of triply charged NZF-domain peptides from TAB2, HOIL-1L-V195R or Sharpin-S346R purified from infected 293T cells. The unmethylated peptides are shown in blue trace and the methylated ones are in red with the methylated cysteine residue in red. The V195R and S346R mutations were introduced to facilitate mass spectrometry identification of the tryptic peptides. C_ca, carbamidomethylated cysteine generated from iodoacetamide treatment during sample preparation; C_me, NleE-methylated cysteine.