FIGURE 3.
Analysis of NADase activity of AbTir-TIR proteins purified by nickel bead chromatography.
(A) SDS-PAGE analysis of recombinant AbTir-TIRWT, S140A, D167A, and W204A proteins expressed in lysates (lys) and purified and eluted (Elu) from a Ni-NTA column with 0.2 M imidazole. (B) Analysis of NADase activity of purified AbTir-TIR proteins. Purified AbTir-TIR proteins were incubated with 5 µM NAD for 1 h at room temperature, and remaining NAD levels in the sample were measured. Means and SDs from three replicates are presented and analyzed by one-way ANOVA (p < 0.0001), followed by Tukey’s post hoc analysis. Asterisks indicate statistically significant differences compared with NAD levels remaining upon incubation with Ab-TIRWT (**p < 0.0001).