Nadh n8129

PPP (HY-15494), GDC-0994 (HY-15947), and ARQ-092 (HY-19719) were purchased from MedChemExpress. Proline (P0380), NAD⁺ (N3014), and NADH (N8129) were purchased from Sigma-Aldrich.

The activity of acyl‐CoA synthetase (ACS) was measured by a continuous coupled enzymatic assay as previously described.^[47] The reaction mixture contained 100 mM Tris‐HCl buffer (pH 8.0), 10 mM ATP (10519979001, Sigma‐Aldrich), 15 mM MgCl₂, 5 mM dithiothreitol, 150 mM KCl, 1 mM potassium phosphoenolpyruvate (P7127, Sigma‐Aldrich), 0.3 mM nicotinamide adenine dinucleotide (NADH, N8129, Sigma‐Aldrich) in 100 mM triethanolamine (pH 8.2), and 500 µM sodium palmitate (P9767, Sigma‐Aldrich). 4.5 µg adenylate kinase (M3003, Sigma‐Aldrich), 3 µg pyruvate kinase (P1506, Sigma‐Aldrich), 3 µg lactate dehydrogenase (L8080, Solarbio), and 3 µg mitochondrial protein sample were added in a total reaction volume of 100 µL. The reaction system was incubated at 37 °C for 1 min, and the reaction was initiated by the addition of CoA (final concentration 600 µM) (C4282, Sigma‐Aldrich). Changes in absorbance of the reaction system at 334 nm were measured every 10 s for 15 min with a recording spectrophotometer (Thermo Fisher Scientific). The reaction rate was calculated using the slope and intercept created from a NADH standard curve.
Total cellular and mitochondrial PKA activities were measured by the nonradioactive PKA Kinase Activity Assay Kit (ab139435, Abcam) following manufacturers' instructions.