GFP:INCENP binds to GST-Cep55 through Mklp2. (A) GFP-Trap kinase assay from BE cell extracts using histone H3 as substrate. GFP-associated histone H3-Ser10 phosphorylation (pH3-Ser10), immunoprecipitated Aurora B, and GFP were analyzed by Western blotting. Histone H3 was analyzed by Ponceau staining. 300 nM VX-680 was added to the kinase reaction to inhibit Aurora B catalytic activity. (B) Cep55:GFP colocalizes with INCENP at the midbody center in BE cells. (C–F) Localization and mean intensity of INCENP at the midbody or midbody center in BE cells. Mean ± SD from n cells. Values in control were set to 1. ***, P < 0.001 (Student’s t test). Tubulin values indicate midbody thickness. Insets show 1.6× magnification of the midbodies. Scale bars, 5 µm. (G) Immunoprecipitation from cytokinesis-enriched BE cell extracts. (H) Predicted experimental outcome. In the Western blot, the width of the lines indicates band signals intensity. (I and J) Cell lysates from BE cells were incubated with GST-Cep55 or GST. Associated proteins were detected by Western blotting. (K and N) Radiolabeled FL or truncated Mklp2 (ΔC90) was incubated with GST-Cep55 or GST. Phosphorimager (35S, top) and Western blot analysis (WB; bottom) of the same gel. Asterisks, predicted molecular weights. (L and M) GFP-Trap assay. Precipitated (L) or input proteins (M) were analyzed by Western blotting. S, serine.
