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. 2010 Sep 6;190(5):793–805. doi: 10.1083/jcb.201002055

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© 2010 Ray et al.

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Figure 2. — The SIN has a dual role in regulation of the Orb6 kinase activity. (A) Ectopic activation of the SIN in interphase inhibits Orb6 kinase activity. All strains expressed mob2-13Myc from the chromosomal promoter. Cells were grown at 25°C, then shifted to 36°C for 3 h. Cell extracts were prepared and the Orb6–Mob2–13Myc complex was immunoprecipitated using anti-Myc antibodies. Immunoprecipitates were split, with one half immunoblotted with anti-Myc antibodies and the other half used to assay Orb6 kinase activity using MBP as an artificial substrate. The kinase activities (KA) were quantified using ImageJ software and normalized to the amount of Mob2–13Myc (IP) and the background kinase activity in untagged control cells. The activity relative to wild-type cells is shown. Error bars denote SD from three separate experiments. (B) The SIN inhibits Orb6 kinase activity in mitosis but promotes its full activity in the following interphase. Early G2 cells of the indicated strains expressing Mob2-13Myc from the chromosomal locus were grown at 25°C, synchronized by elutriation, and then shifted to 36°C at time zero, and portions were collected at the indicated times and assayed for Orb6 kinase activity. This experiment was repeated at least once with similar results.