Fig. 3.

CD38 can support BAC synthesis in intact cells. (A) Total membrane fractions from control (CD38⁻) and CD38⁺ HeLa cells were incubated for 2 h at 37 °C with recombinant His-BARS and 30 µM total NAD⁺ (spiked with 5 µCi [³²P]-NAD⁺), in the absence and presence of BFA (80 µg/mL). The samples were analyzed by SDS/PAGE and autoradiography (AR [³²P]); total BARS levels were analyzed by Western blotting. (B) CD38⁺ HeLa cells were transfected with wild-type YFP-BARS (BARS WT) or YFP-BARS with the His304 point mutation (BARS H304A). After 24 h, the cells were treated with 80 µg/mL BFA for 4 h at 37 °C, in the presence of 5 mM extracellular NAD⁺. YFP-BARS was immunoprecipitated using an anti-BARS antibody, and the modified protein was revealed using an anti–BFA-specific antibody. (B, Lower) Total BARS levels are shown. (C) CD38⁺ HeLa cells were transfected with YFP-BARS. After 24 h, the cells were treated with 80 µg/mL BFA for 1 h or 4 h at 37 °C, in the presence or absence of exogenously added 5 mM NAD⁺, as indicated. YFP-BARS was immunoprecipitated from total lysates using an anti-BARS antibody, and the modified protein was revealed using an anti–BFA-specific antibody. (C, Lower) Total levels of BARS are shown.