FIGURE 9.

Mitotic spindles were not assembled properly with the phospho-resistant mutant of centrobin. A, HeLa cells were transfected with siCBN and subsequently with the wild type (pGST-MycCBN) or phospho-resistant (pGST-MycCBN^{T3A,S4,21,22A}) centrobin plasmids. The endogenous and ectopic (WT, GST-MycCBN, and MU, GST-MycCBN^{T3A,S4,21,22A}) centrobin protein levels were determined by immunoblot analysis with the centrobin antibody. β-Tubulin was detected as a loading control. B, 48 h after transfection, the cells were treated with monastrol (100 μm) for 6 h and released for 1.5 h in the presence of MG132 (10 μm). The cells were coimmunostained with antibodies specific to Myc and γ-tubulin. DNA was stained with 4′,6-diamidino-2-phenylindole. The spindle morphology was classified into bipole, abnormal bipole, and monopole. The experiments were repeated five times and 100 cells per experimental group were analyzed. The results are indicated as mean ± S.D.