Figure 7.

Phosphorylation of XMAP4 and mutants in mitosis. (A) Mobility shift of XMAP4 and mutants in mitosis. Interphase (I) and mitotic (M) extracts of A6 cells expressing GFP–WT, GFP–CM10A, GFP–C6A, GFP–CM8A, or GFP–R4A were immunoblotted with anti-XMAP4 antibody. Arrowheads and arrows denote endogenous XMAP4 and GFP–XMAP4, respectively (see Figure 3). Note that the bands of GFP–WT and GFP–R4A as well as endogenous XMAP4 were shifted upward up to the top arrow and arrowhead in mitosis, whereas those of GFP–CM10A, GFP–C6A, and GFP–CM8A did not show any mobility shift. (B) Mobility shift of XMAP4 by phosphorylation. Heat-stable XMAP4 fractions were prepared from interphase (I) and mitotic (M) A6 cells expressing GFP–WT. The fractions were treated with alkaline phosphatase in the presence or absence of the phosphatase inhibitor β-glycerophosphate (30 mM) and then immunoblotted with anti-XMAP4 antibody. (C) XMAP4 phosphorylation by p34^cdc2 kinase. Heat-stable XMAP4 fractions were prepared from interphase (I) and mitotic (M) A6 cells expressing GFP–WT or GFP–CM10A and then phosphorylated by p34^cdc2 kinase (+). Immunoblotting was with anti-XMAP4 antibody. Arrowheads and arrows denote endogenous XMAP4 and GFP–XMAP4, respectively.