AMPK phosphorylates NADSYN1 at Serine 641. (A) Schematic of AMPK phosphorylation and transactivation of NADSYN1. (B) Bottom panel represents in vitro kinase assay using (γ-32P) ATP, full-length recombinant NADSYN1 without (left) and with recombinant (right), active AMPK. Top panel represents to Coomassie blue (CB) stain to demonstrate equal substrate protein loading. Scintillation counter per minute (CPM) quantification of kinase assay repeated using NADSYN1 Ser641 and Ser641A peptides (C) or full length NADSYN1 (D). Background represents reactions with only (γ-32P) ATP without AMPK nor substrate. AMPK represents reactions with AMPK, (γ-32P) ATP, but no substrate. SAMS is a peptide containing the AMPK phosphorylation site in acetyl CoA carboxylase, serving as a positive control. Student t-test used to determine *p < 0.5, all experiments were repeated at least 3 times.