Figure 4

Phosphorylation of Borealin in mitosis. Hela cells were transfected with Flag-Borealin that was analyzed by Western blotting with an antibody to the Flag-tag and endogenous Borealin. (A) Mitotic phosphorylation of Borealin. Hela cells transiently transfected with the T106A mutant of Borealin were either allowed to grow asynchronously or blocked in mitosis using nocodazole. Lysates from transfected, mitotic cells were treated with phosphatase for 1 hrs (PP1) and 4 hrs (PP4) in vitro and also with phosphatase inhibitor (PP+Inh). Cell lysates were run on a 15% or 12.6% SDS-polyacrylamide gel. * : Nocodazole treated cells, only the upper, shifted form of Flag-tagged and endogenous Borealin is observed. (B) WT-8 cells were blocked in mitosis using nocodazole and then released into fresh medium. Cells were harvested at the indicated time points and cell lysates were separated by a long run on a 12.6% SDS-polyacrylamide gel. Membranes were stripped and reprobed for Cyclin B1 as a marker for progression through the cell cycle and for β-actin as a loading control.