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Fig. 5. Eg5[Ser1033] phosphorylation is required for normal Eg5 function in mitotic spindle assembly. (A) U2OS cells transiently expressing Myc-Eg5 wild type, Myc-Eg5[Thr926Ala] or Myc-Eg5[Ser1033Ala] were stained for Myc, β-tubulin and DNA. A representative transfected cell in mitosis is shown in each case, except for Myc-Eg5[Ser1033Ala], for which a mitotic cell plus two transfected interphase cells is shown. (B) HeLa cells were co-transfected with either control oligonucleotides (lanes 1-6) or siRNA oligonucleotides directed against human Eg5 (lanes 7-12), together with empty plasmid (lanes 1 and 7) or plasmids encoding Myc-Eg5 wild type (wt) that was unmodified (lanes 2 and 8) or Myc-Eg5 variants that were rendered RNAi resistant (rrEg5 wt, Eg5 wild type; rrEg5 TA, Eg5[Thr926Ala]; rrEg5 SA, Eg5[Ser1033Ala]; rrEg5 SD, Eg5[Ser1033Asp]). Cell lysates were immunoblotted with anti-Myc (upper panel) or anti-Eg5 (middle panel) to determine recombinant and total Eg5 expression, respectively, and with anti-
-tubulin antibodies to evaluate loading (bottom panel). (C) HeLa cells were co-transfected with siRNA oligonucleotides directed against human Eg5 and with plasmids encoding Myc-GFP (control) or several RNAi-resistant Myc-Eg5 (rrEg5) variants. At 48 hours after transfection, cells were fixed and stained with anti-Myc and anti-β-tubulin antibodies, and with DAPI. Myc-positive cells were scored as being in interphase (gray columns), in normal mitosis (white columns) or in an abnormal mitosis (mostly monopolar spindles; black columns). The figure represents the mean of three independent experiments, wherein >100 cells were scored for each point in each of the experiments; error bars indicate s.e.m. The fraction of abnormal mitoses in cells expressing rrEg5 wild type was compared with each of the other conditions by Student's t-test; *P<0.05. The comparison of Ser1033Ala with Ser1033Asp (S1033D) gave a P value of 0.068.
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