Mitotic phosphorylation of the cytoplasmic dynein ICs. (A) SDS-PAGE analysis of HeLa cell cytoplasmic dynein. Dynein ICs (arrows) displayed a gel shift in mitotic but not interphase samples, and the gel-shifted form was eliminated by λ-phosphatase treatment. (B) Western blot analysis of dynein ICs at time points after nocodazole washout. Arrows indicate two variants of the dynein ICs. (C) Blot overlay analysis of interphase and mitotic ICs using recombinant p150Glued. Controls confirm equivalent loading of interphase (I) and mitotic (M) dynein ICs. Overlay assays reveal reduced binding to mitotic ICs compared with interphase ICs. The signal of the interphase sample was scored as 100%, and the signal of the mitotic sample was measured as a percentage of the interphase sample after background subtraction (n = 3; P = 0.007). Error bar represents SD. (D) Gel-shifted IC gel bands were subjected to in-gel tryptic/chymotryptic digestion and microcapillary liquid chromatography tandem MS analysis. Collision-induced dissociation identified the phosphorylated residue as T89 (Fig. S1 A, available).
