Phosphorylation of Borealin during interphase. Mobility shift of Borealin was analyzed by Western blotting using a 12.6% SDS-PAGE. Western transfers were stripped and reprobed for β-actin as a loading control. UT : Untransfected. (A) Effect of cyclohexamide (CHX) on asynchronous cells. Asynchronously growing WT-8 cells were treated with CHX for the indicated times. (B) Effect of CHX on cells blocked in S phase. WT-8 cells blocked in S phase by treating with hydroxyurea for 16 hours were exposed to CHX or water (vehicle) for 2 hours (in the continuous presence of hydroxyurea). (C) WT-8 cells were either allowed to grow asynchronously or blocked in S phase using hydroxyurea for 16 hours and then treated with CHX for 2 hours. Cell lysates were treated with phosphatase for 1 hr (PP1) and 4 hrs (PP4) in vitro and also with phosphatase inhibitor (PP+Inh).