FIGURE 5.

PP2A and p27. A, 500 μg of extract from 293T cells transfected with HA-tagged versions of the different B56 isoforms, indicated above each lane, was immunoprecipitated (IP) with anti-HA, and the presence of p27 and cyclin G was determined by immunoblot (IB). This image is representative of two independent transfections. The expression of the B56 isoforms was determined in the top panel by immunoblotting. B, PP2A can dephosphorylate serine 10 and threonine 187. Top panel, [³²P]-labeled recombinant His-tagged human p27 was incubated with increasing amounts of purified PP2A. Reactions were stopped by the addition of sample buffer, and proteins were separated by SDS-PAGE followed by autoradiography. This experiment was repeated twice with similar results. Bottom panel, the experiment was performed as above except that cold ATP was used instead of radiolabeled ATP and phosphorylation was detected by immunoblotting with phospho-specific antibodies to Ser-10-phosphorylated p27 (pS10-p27) or Thr-187-phosphorylated p27 (pT187-p27). This experiment was repeated twice with similar results. C, NIH3T3 cells were serum-starved for 24 h and infected in the absence of serum with either an empty adenoviral vector (CMV) or one expressing ST. The cells were harvested 24 h later, and the expression of ST, p27, cyclin A (cycA), and the Ser-10-phosphorylated(pS10-p27) and Thr-187-phosphorylated p27 (pT187-p27) isoforms was measured by immunoblotting. This image is representative of three independent experiments. MOI, multiplicity of infection.