FIG 6.

Direct cleavage of p38 by NleD in vitro and mass spectrometric analysis of putative cleavage site for p38. (A) Immunoblots of GST or GST-p38 incubated with His-NleD or His-NleD site-directed mutants. Arrows, p38 cleavage fragment. Reaction mixtures were probed with anti-GST antibodies, and anti-His antibodies were used to detect NleD and NleD variants. (B) A Coomassie-stained gel showing the N-terminal (N-term) and C-terminal (C-term) cleavage products of GST-p38 in the presence of His-tagged NleD after incubation for 2 h or overnight (O/N). White boxes, bands that were excised from the gel and used for further mass spectrometric analysis. (C) Identification of semitryptic peptide ²¹³MAELLTGR²²⁰ only within the C-terminal p38 cleavage fragment supports the suggestion that cleavage of p38 occurs at or before position 213. M_ox, oxidized methionine residue; a, b, and y, peptide fragment ions.