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. 1999 Nov;10(11):3927–3941. doi: 10.1091/mbc.10.11.3927

Figure 2.

Figure 2

A protein phosphatase of type 2A is required to maintain cyclin degradation activity in G1. (A) HeLa cell G1 extracts were preincubated with various kinase inhibitors (20 nM staurosporine, 5 mM 6-DMAP, 50 μM olomoucine) or phosphatase inhibitors (inhibitor mix, see MATERIALS AND METHODS; 1 mM sodium orthovanadate, 1 μM OA, 200 nM heatstable inhibitor I-2) as indicated before the addition of radiolabeled cyclin B protein and assayed as in Figure 1. (B) Histone H1 kinase activity in Hela cell G1 extracts treated with kinase or phosphatase inhibitors or both. Hela cell G1 extracts preincubated with DMSO, 20 nM staurosporine (Stsp), 1 μM OA, or 1 μM OA plus 20 nM stauroporine were subjected to histone H1 kinase assays. Phosphorylated histone H1 was resolved on 20% SDS-PAGE followed by autoradiography. (C) OA does not activate a kinase activity that is responsible for inactivating the cyclin B degradation activity in G1 extracts. HeLa cell G1 extracts used in B were preincubated with DMSO (as a control), 20 nM staurosporine (Stsp), 1 μM OA, or 1 μM OA acid plus 20 nM staurosporine before assaying the cyclin B degradation activity as decribed previously.