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. Author manuscript; available in PMC: 2010 May 1.
Published in final edited form as: Nat Cell Biol. 2009 Apr 26;11(5):644–651. doi: 10.1038/ncb1871

Figure 2. PP1 is required for the dephosphorylation of substrates at mitotic, but not meiotic exit.

Figure 2

A. Ros (0.28mM) was added to CSF extracts in the presence of GST or GST-I2. Aliquots were withdrawn at the indicated times and immunoblotted with anti-Cdc27, anti-pPlx, or MPM2 antibody. Full scans of Cdc27 and pPlx blots are shown in S. Fig. 5B.

B. Ros (0.28mM) was added to CSF extracts in the presence of GST or GST-I1. Aliquots were withdrawn at the indicted times for Cdc27 immunoblotting and HH1 kinase assays.

C. Left: PP1 was immunodepleted from mitotic extracts using anti-Xenopus PP1 antibodies. Rabbit IgG was used for mock depletion. Control or PP1-depleted extracts were treated with Ros (0.28mM). Aliquots were immunoblotted with anti-Cdc27 antibody. Right: Control and PP1-depleted extracts were immunoblotted with PP1 antibodies.

D. Left: Same as Figure 2C except that CSF extract was used for PP1 depletion. PP1 depleted extract was supplemented with buffer or recombinant His-PP1 (0.5 μM). In addition to Cdc27 immunoblotting, HH1 kinase activity was assayed. Right: Extracts were also immunoblotted with PP1 antibodies. Full scan of PP1 blot is shown in S. Fig. 5C.

E. GST or thio-phosphorylated GST-I1 was added to cycling extracts and aliquots were withdrawn at the indicated times and immunoblotted with anti-Cdc27 and anti-Cyclin B2 antibodies. Arrows indicate gel mobilities of Cdc27. Full scan of Cdc27 and Cyclin B2 blots are shown in S. Fig. 5D.

F. HeLa cells were arrested in Mitosis with Nocodazole for 17 hours, and then allowed to resume cell cycle in fresh DMEM for 1 hour. Cell lysates were made and supplemented with GST or GST-I1. Aliquots were taken to immunoblot with MPM2 antibody and to measure HH1 kinase activity.

G. Ca2+ was added to CSF extracts in the presence of GST or GST-I1. Aliquots were immunoblotted with anti-Cdc27 antibody.

H. Left: PP1 antibodies were used for PP1 immunodepletion and rabbit IgG was used for control depletion. Calcium was added to the depleted CSF extracts. Aliquots were taken at the indicated times and blotted with anti-Cdc27 antibody. Right: Aliquots of depleted extracts were blotted with anti-PP1 antibody.