A, human CTP synthetase 1 (50 µg/ml) was phosphorylated with mammalian protein kinase C (0.5 unit/ml) and 50 µM [γ-32P]ATP (11,000 cpm/pmol) for 20 min followed by SDS-PAGE, transfer to polyvinylidene difluoride membrane, and phosphorimaging analysis. B, the PVDF membrane slice containing 32P-labeled enzyme was hydrolyzed with 6N HCl for 90 min at 110 °C, and the hydrolysate was separated by 2-dimensional electrophoresis. The positions of the standard phosphoamino acids phosphoserine (P-Ser), phosphothreonine (P-Thr), and phosphotyrosine (P-Tyr) are indicated in the figure. The data shown are representative of three independent experiments.