TPX2 interacts with CLASP1 in a phospho-dependent manner, and its function in scaling spindle is bypassed by codepletion of CLASP1 and Kif2a. (A) Mitotic HeLa cells transfected with the indicated constructs were immunoprecipitated with TPX2 antibody. Kif2a and CLASP1 form complexes with TPX2, whereas CLASP2 and GFP could not. (B) Mitotic HeLa lysates were immunoprecipitated with IgG, CLASP1, or Kif2a antibody. CLASP1 and Kif2a specifically bind to TPX2. The asterisk indicates Kif2a. (C) Mitotic cells transfected with GFP-tagged TPX2 constructs were immunoprecipitated with CLASP1 antibody. TPX2-2A binds significantly less CLASP1 than -WT and -2D. (D) Mitotic cells cotransfected with GFP-Kif2a and Flag-tagged TPX2 constructs were immunoprecipitated with flag antibody. TPX2, 2A, and 2D show similar affinity to Kif2a. (E and F) HeLa cells co-depleted of CLASP1/Kif2a were transfected with GFP-TPX2 or -2A. In mock-depleted cells, overexpression of TPX2-2A causes short spindles whereas in CLASP1/Kif2a-depleted cells, it does not affect the spindle length. Bar, 10 µm. n = 81, 89, 73, and 86. Error bars indicate SEM. ***, P < 0.001.