Kinase experiment:

For Cdc5 kinase assays, Spc72-TAP is isolated from cell extract by pulldown with IgG sepharose or rabbit IgG coupled to M-270 epoxy dynabeads and incubated in kinase buffer (25 mM HEPES, pH 8.0, 60 mM KCl, 15 mM MnCl2, 100 μg/mL BSA, 80 nM microcystin, 1mM DTT, 100 μM 200 μCi/mL [γ-32P]ATP) in the absence or presence of 100 ng purified baculovirus expressed 6xHis-Cdc5. 32P incorporation is visualized on a Typhoon PhosphorImager, and images are processed using ImageQuant software.

CMK is a RSK2 kinase inhibitor which exhibits similar potency but less chemical stability compared with FMK.

CMK inhibits the growth of Cdc5 (L158G) with IC50 of 36 nM, greater than 30 μM for wild type Cdc5. CMK exhibits a concentration-dependent first cell cycle mitotic arrest in the cdc5-as1 strain with an IC50 of 1.1 μM. CMK inhibition of Cdc5 (L158G) leads to a first cell cycle anaphase arrest and delay in anaphase spindle migration[1].

References:
[1]. Snead JL, et al. A coupled chemical-genetic and bioinformatic approach to Polo-like kinase pathway exploration. Chem Biol. 2007 Nov;14(11):1261-72.