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Fig. 5. (A) Cdc2 kinase activity and protein levels were assayed in cdc37-681, cdc37-184 and cdc37⁺ cells after growth at 28 and 36°C. Strains were cultured at 28 and 36°C over a 3 hour time course. Samples of cells were taken hourly and Cdc2 was affinity-precipitated on p13^Suc1 beads. The kinase activity of Cdc2 was determined by its ability to phosphorylate histone H1. Cdc2 protein levels were determined by western blot with the anti-PSTAIR antibody and ß-tubulin detected by TAT1 antibodies as a loading control. The asterisk indicates the position of p31 which is also recognised by the anti-PSTAIR antibody (see text). (B) The level of Cdc2 protein in soluble and insoluble fractions of extracts of cdc37-184, cdc37-681 and cdc37⁺ cells grown at 28°C or incubated at 36°C for 3 hours was analysed. Native protein extracts were prepared and the soluble and insoluble fractions separated by centrifugation at 20,000 g for 5 minutes at 4°C. Western blot analysis with anti-PSTAIR antibody against Cdc2 and TAT1 antibody as a loading control. (C) Level of phosphorylation on Tyr15 of Cdc2 in cdc37-184, cdc37-681 and cdc37⁺ strains incubated at 28 and 36°C over a 3 hour time course. Samples of cells were taken hourly and denatured S. pombe proteins extracted and western blotted with antibody specific for Cdc2 phosphotyrosine 15.

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