Label-free pull-downs of TACC3 untreated and treated with aurora A inhibitor. (A and B) Live imaging of cells expressing GFP-tagged TACC3 and mCherry-tagged α-tubulin. DNA was stained with Hoechst. (top) Chromosome alignment and spindle morphology are shown. (bottom) The fluorescence signal of GFP-tagged TACC3 is shown. (A) TACC3^WT normally localizes to spindles in untreated cells (left) but is mislocalized away from spindles after treatment with the aurora A kinase inhibitor MLN8054, similar to TACC3^AAA (middle and right). Under both MLN8054-treated and mutant TACC3 conditions, spindle morphology and chromosome alignment are compromised. (B) The RNAi-resistant TACC3^AAA mutant does not localize to spindles after RNAi of endogenous TACC3 (middle and right). (C) Volcano plot representing differential binding partners of TACC3 in dependence of treatment with aurora A kinase inhibitor. The logarithmic ratios of protein intensities are plotted against negative logarithmic p-values of the t test performed from triplicates. Proteins binding specifically in either condition are marked in black and annotated. (D) Localization of TACC3 after RNAi of phospho-dependent interactors. Cells expressing TACC3^WT and mCherry–α-tubulin were transfected with control (CON), CLTC, or GTSE1 siRNAs, and live cells were imaged after 72 h. TACC3 is mislocalized from spindles after CLTC but not GTSE1 RNAi. Bars, 10 µm.