FIGURE 3.

CDK1 mediates the phosphorylation of Ajuba at Ser¹¹⁹ and Ser¹⁷⁵ during G2/M phase arrest. A, HeLa cells were treated with Nocodazole for 8 h and then fixed. Before the cells were stained with phospho-specific antibody against Ser¹¹⁹ of Ajuba, the cells were preincubated with PBS (no peptide control), or non-phosphorylated (control) peptide, or the phosphorylated peptide used for immunizing rabbits. CDK1 inhibitors RO3306 (5 μm) or Purvalanol A (10 μm) together with MG132 (25 μm) were added 2 h before the cells were fixed (bottom two rows). B, experiments were done similarly as in A with phospho-specific antibody against Ser¹⁷⁵ of Ajuba. C, HeLa cells were treated and stained with phospho-specific antibodies as in A and B. A ×63 oil objective lens was used to view fewer cells in a field. P-H3 S10 was used as a mitotic marker. White and yellow arrows mark some of the prometaphase cells and the interphase cells, respectively.