FIGURE 2.

ISTIDs of human NMNATs are dispensable for catalytic activity. The NMNAT activities of ISTID deletion mutants (Δ, ○) NMNAT1_ΔISTID1 (A), NMNAT2_ΔISTID2 (B), and NMNAT3_ΔISTID3 (C) overexpressed in HeLa S3 cells were determined and compared with those of the respective wild-type proteins (wt, ●). The use of comparable amounts of expressed protein was verified by Western blotting as shown. Extracts from non-transfected cells were used as controls in both activity measurements (×) and Western blots (c). Reactions were started by the addition of NAD⁺ (arrows). All experiments were performed at least twice in duplicates. Representative data sets are shown. RLU, relative light units. Note that purified recombinant human NMNATs exhibit relative catalytic activities of ∼20:1:2 (NMNAT1/NMNAT2/NMNAT3) (27).