C4282

CoA-agarose (C7013-500MG, Sigma-Aldrich) beads were suspended in 3.5 ml of 10 mM sodium acetate (pH 6.0) and stored at −20°C. For each experiment, 50 μl of the CoA-agarose mixture was washed three times in lysis buffer (250 mM KCl) and mixed with 1 ml of each of the lysates (respectively from testis, spermatogenic cells, or liver) and incubated with rotation overnight at 4°C. The CoA-agarose beads were then washed three times in the lysis buffer, followed by a final wash with PBS. Half of the beads were then incubated for 1 hour at 4°C in 50 μl of 5 mM free CoA (C4282, Sigma-Aldrich) (dissolved in H₂O), and the other half was incubated in 50 μl of H₂O (Control). Eluted proteins were collected following a centrifugation step (1650g, 2 min, 4°C). Ten microliters of the eluted proteins was separated by migration on a NuPAGE 4 to 12% bis-tris protein gel (Invitrogen) and silver-stained. Another 10 μl was analyzed by Western blotting (WB). The rest of the eluted proteins were analyzed by MS.

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.