The CHC–GTSE1 interaction recruits GTSE1 to the CHC/TACC3 complex and the spindle but does not control TACC3 spindle localization. (A) Immunoblot on mitotic lysates of GTSE1^WT and GTSE1^5xLID cells (Input) and IPs of the GFP transgene (GFP IP). MLN8054 was used to inhibit Aurora A. Immunoblots are with anti-GFP, anti-CHC, or anti-TACC3. (B) GST and GST-GTSE1 pulldowns of purified TACC3 phosphorylated by Aurora kinase A. GST-GTSE1 was left unphosphorylated or was phosphorylated by Aurora A (Aur. A), Aurora B (Aur. B), Cdk1, or Aurora A, Aurora B, and Cdk1 (ABCdk1). Phosphorylated TACC3 and GTSE1 were visualized by immunoblot against pTACC3(S558) and GTSE1, respectively. (C) SDS-PAGE stained with Coomassie blue of GST, unphosphorylated GST-GTSE1, and GST-GTSE1 phosphorylated by Aurora A (Aur. A), Aurora B (Aur.B), Cdk1, or Aurora A, Aurora B, and Cdk1 (ABCdk1). Presence of phosphorylated residues was confirmed by Pro-Q staining. (D) Still images from live-cell imaging on GTSE1^WT and GTSE1^5xLID cells transfected with GTSE1 siRNA to deplete the endogenous GTSE1. Cells were treated with siRTubulin before imaging to visualize MTs. (E) Immunofluorescence images showing the localization of TACC3 to the spindle in GTSE1^WT and GTSE1^5xLID cells transfected with the GTSE1 siRNA. TACC3 and α-tubulin staining are shown. (F) Quantification of TACC3 on the spindle of cells from E. Mean TACC3 fluorescence intensity on the spindle was corrected for background and normalized to α-tubulin (n ≥ 51 cells per condition over n = 3 experiments pooled for analysis and presented as a box and whisker plot; P value from ANOVA). n.s., not significant. All scale bars: 5 µm. Numeric data is shown in Table S1.