14c nad

Deacylation reactions were performed as described for SIRT3 [10 (link)] and SIRT5 [7 (link)]. In some experiments deacylation was followed as the cleavage of ¹⁴C-NAD⁺ (Perkin Elmer, Waltham, MA) to ¹⁴C-NAM as described [10 (link)] with a reaction time of 20 minutes at 37°C.

Cells plated in 12 wells plates were grown to confluence. When cells were preincubated with 1 mM unlabeled NAD+ for 24 h, the wells were washed three times with 1 ml of PBS and then incubated in a buffer solution (140 mM NaCl, 10 mM Hepes/Na, 2.5 mM MgSO₄, 2 mM CaCl₂, and 5 mM KCl, 5 mM glucose, pH 7.4) containing 500 pM [¹⁴C]NAD+ (54 mCi/mmol), [³H]NAD+ (28.6 Ci/mmol), or [¹⁴C]AMP (60 mCi/mmol) (PerkinElmer). Competing compounds and drugs were added to the wells 15 min before labeled molecules unless stated in the text. After 10 min, cells were washed three times with 1 ml of PBS and lysed with 0.5 M NaOH. Incorporated radioactivity was assayed by liquid scintillation counting.