Axon-protective function of NAD⁺-producing enzymes is specific to Nmnat1 and -3, and Nmnat3 protects as well as Wld^S. (A) NADS and N16-NADS were expressed in ORNs using OR22a-Gal4, and axons were severed and analyzed for protection 5 d after axotomy. (B) Localization of mCD8-Wld^S was assayed using α-CD8 antibodies, and expression of mCD8-Wld^S was driven in ORNs using the OR22a-Gal4 driver. Axons are indicated projecting to glomeruli (arrowhead) and across the midline (arrow), and OR22a⁺ ORN-innervated glomeruli are shown with dashed lines. (C) Membrane-tethered Wld^S (mCD8-Wld^S) was assayed for axon-protective function 10 d after axotomy. n ≥ 10 for all genotypes. (D) Nmnat2 and -3 were driven by OR22a-Gal4 in ORNs to assay for protective function 5 d after axotomy. n ≥ 10 for all genotypes; **, P < 0.01; ***, P < 0.001. (E) S2 cells were transfected with Nmnat2-myc and Nmnat3-myc and stained with α-myc. In both cases, α-myc immunoreactivity was localized to the cytoplasm but not the nucleus. DIC, differential interference contrast. Error bars represent SEM. Bars: (B) 27.47 µm; (E) 14.71 µm.