FIGURE 4.

Increased nicotinamidase activity in transgenic Drosophila lines overexpressing D-NAAM. A, expression of V5-D-NAAM protein in pUAST-D-NAAM transgenic lines in tubulin-Gal4 driver background was analyzed using V5 immunoblotting. B, D-NAAM protein expression in control w¹¹¹⁸ flies, a high expressing D-NAAM transgenic line 42am (tubulin-Gal4 driver), S2 cells, and control COS-7 cells were analyzed using a carboxyl-terminal D-NAAM antibody. C and D, nicotinamidase activity in protein extracts of the indicated fly lines or S2 cells was analyzed by immunoprecipitating (IP) D-NAAM using V5 or a carboxyl-terminal D-NAAM antibody and assaying amidase activity as in Fig. 2B. Preimmune serum for the D-NAAM antibody (PS) was used as a control antibody. The difference seen in activity between V5 and D-NAAM immunoprecipitates reflects the difference in immunoprecipitation efficiencies (data not shown). Note that the low activity seen in the w¹¹¹⁸ sample is a result of assay linearity limitations (the total amount of protein to be used for the immunoprecipitation was determined in a way to allow remaining in the linear range of the assay for the high expressing D-NAAM lines). NAM, nicotinamide.