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. 2008 Jul;19(7):2907–2915. doi: 10.1091/mbc.E07-07-0679

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© 2008 by The American Society for Cell Biology

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Figure 3. — B-Raf activity in M-phase egg extracts is coupled to Cdk1 activity. (A) Time course of Cdk1/cyclin B and B-Raf activation after GST-Δ90 cyclin B addition to S-phase–arrested Xenopus egg extracts. B-Raf was IPed from aliquots collected at the indicated time points and subjected to an in vitro–linked kinase assay. Equal loading of B-Raf immunoprecipitates per kinase assay was confirmed by B-Raf Western blotting (not shown). Cdk1/cyclin B activity was measured in in vitro histone H1 kinase assay. M-phase was monitored by nuclear envelop breakdown of nuclei formed from added sperm DNA. (B) Cdk1/cyclin B activity is necessary for B-Raf activation during mitosis. Cdk1 activity in M-phase–arrested extracts was inhibited with 10 μM Flavopiridol. B-Raf was immunopurified from S-phase, M-phase, and M-phase extracts treated with Flavopiridol to assess its activity by an in vitro–linked kinase assay. Equal loading of B-Raf immunoprecipitates per kinase assay was confirmed by B-Raf Western blotting. Cdk1/cyclin B activity was measured in in vitro histone H1 kinase assay.