The MBD of p150^glued is phosphorylated by aurora A after nuclear envelope breakdown. (A) Wild-type (WT) or mutant MBD fragment in which the eight Sers (phosphorylated by aurora A) were mutated into Alas (MBD-SA8) were subjected to a kinase assay in the presence of active (AUR A WT) or inactive (AUR A K/R) recombinant aurora A kinase. The Coomassie blue (CB)–stained gel (top) was subjected to autoradiography (bottom). The p150^glued MBD fragment (closed arrowhead) is strongly phosphorylated in the presence of active (but not inactive) aurora A protein kinase, whereas the MBD-SA8 fragment (open arrowhead) remains unphosphorylated. (B) Scheme of the wild-type (MBD-GFP) or mutant (MBD-SA8-GFP) proteins stably expressed in S2-cultured cells. (C) Control (cont) or S2 cell extracts expressing MBD-GFP or MBD-SA8-GFP were separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and stained by Ponceau S as a loading control (left). The membrane was blotted for p150^glued. The position of the ∼50-kD MBD-GFP and MBD-SA8-GFP proteins is indicated (arrowhead). (D–F) Interphase S2 cells expressing MBD-GFP (top) or MBD-SA8-GFP (bottom) were fixed and stained for tubulin (red; middle in monochrome) or GFP (green; right in monochrome). Both GFP fusions associate with the interphase microtubule network. S2 cells expressing either MBD-GFP (E) or MBD-SA8-GFP (F) were methanol fixed and stained for DNA (blue), tubulin (red; middle in monochrome), and GFP (green; bottom in monochrome). During prophase, the GFP signal was strong at the centrosome region for both proteins. Unlike MBD-GFP, the mutant MBD-SA8-GFP protein remains strongly associated with spindle microtubules during all mitotic steps. Bars, 10 µm.