Binding of the CLASP2 plus end tracking domain to the tubulin C terminus and EB1 is directly inhibited by GSK3β phosphorylation. (A) Sedimentation assay of 6×His-CLASP2(340–650) at constant concentration (0.5 µM) with an increasing concentration of taxol-stabilized MTs. Comparison of the WT with a mutated protein in which the GSK3β sites between S594 and S610 were replaced with phosphomimetic aspartate residues (5×S/D) shows that the phosphomimetic mutant does not bind MTs. (B) Immunoprecipitation (IP) using GFP antibodies from HeLa cells expressing EGFP-tagged CLASP2–MT plus end tracking domain constructs. Endogenous EB1 only immunoprecipitates with WT EGFP-CLASP2(512–650) and the nonphosphorylatable mutant 9×S/A but not with pseudophosphorylated 8×S/D or EGFP alone. (C) Sedimentation assay using MTs treated with subtilisin, which removes the flexible tubulin C terminus, resulting in a downshift of the α/β-tubulin bands on the Coomassie-stained gel. 6×His-CLASP2(340–650) does not bind to subtilisin-treated MTs.