Figure 1.

Phosphorylation of PP-1C in vitro and detection by phosphorylation-state-specific antibodies. (A) Sf9 PP-1Cα (Left) and PP-1Cγ (Right) were phosphorylated by purified cdc2/cyclin B (New England Biolabs) and nonradioactive ATP for 90 min. The samples were resolved by SDS/PAGE (10% acrylamide) and immunoblotted with G-97 antibody (anti-T320 phosphorylation-state-specific PP-1Cα antibody). (B) Sf9 PP-1Cα was incubated with cdc2/cyclin B for various times in the presence of [γ-³²P]ATP (Left) or nonradioactive ATP (Right). Samples were resolved by SDS/PAGE. [³²P]-Labeled PP-1Cα was detected by autoradiography. Samples incubated with nonradioactive ATP were transferred to Immobilon-P, followed by immunoblotting with G-98 antibody and autoradiography. The region of the gel or immunoblot containing PP-1C is shown. No immunoreactivity was observed in any other part of the immunoblot.