Fig. 5.
S119 is critical for Mtb PafA depupylase activity.
(a) PanB-PupE (0.75 μM) were depupylated by PafA variants (1.5 μM each) with 5 mM ADP in phosphate buffer for 6 h at 25 °C. Samples were analyzed by SDS-PAGE, followed by Coomassie brilliant blue (CBB) staining or western blotting with an anti-flag antibody. Quantitation of PanB-Flag based on CBB staining is shown in the lower panel. Data are representative of three independent biological replicates (mean and s.e.m. of n = 3 samples), *P < 0.05 and **P < 0.01 (two-tailed unpaired t-test).
(b) As in Fig. 5a, except that the PafA (1.5 μM) pre-incubated with AEBSF at 25 °C for 0.5 h was used instead of PafA variants.