CLASP2 phosphorylation by Plk1 at KTs requires Cdk1-mediated priming. (A) Immunofluorescence localization of pS1255 epitope (green) in mitotic HeLa cells stained for total CLASP2 (red) and DNA counterstained with DAPI (blue). (top) pS1255 staining alone during mitosis. (left to right) KT insets of selected planes from z stacks of the boxed area represent pS1255, CLASP2, ACA, and Merged. Midbody colocalization during telophase is highlighted in the merged figure. (B) Representative immunofluorescence images of pS1255 at KTs upon nocodazole treatment, or combined with Cdk1, Plk1, or both kinase inhibitors. (left to right) Insets show KT single planes of the boxed regions of pS1255 (green) and ACA (red) staining. Bars: 5 µm and 1 µm (higher magnifications). (C) Interquartile representation of pS1255 KT intensities in cells treated with nocodazole (n = 491), nocodazole + Cdk1 inhibitor (n = 459), nocodazole + Plk1 inhibitor (n = 471), and nocodazole + Cdk1 + Plk1 Inhibitors (n = 429). Statistically different values are indicated with *, P < 0.05, Dunn’s Multiple comparison test. (D) CLASP2, pS1234, pS1255, and α-tubulin Western blot of asynchronous and mitotic cells with and without inhibition of Cdk1, Plk1, or both. (E) CLASP2 Western blot analysis of asynchronous and mitotic HeLa cells expressing GFP-CLASP2 (wild-type) or GFP-CLASP2 S1234A.