Label-free pull-downs of TACC3 and TACC3 interaction partners. (A) Live imaging of cells expressing GFP-tagged TACC3 and mCherry-tagged α-tubulin. DNA was stained with Hoechst. The RNAi-resistant TACC3^WT localizes to the spindles in mitosis after RNAi of endogenous TACC3. (left) Chromosome alignment and spindle morphology are shown. (right) The fluorescence signal of GFP-tagged TACC3 is shown. (B–D) Volcano plots representing results of the label-free pull-downs of GFP-tagged TACC3, CLTC, and GTSE1. The logarithmic ratios of protein intensities are plotted against negative logarithmic p-values of the t test performed from triplicates. The hyperbolic curve separates specifically interacting proteins marked in black (red dotted line) from background (blue dots). Names of all proteins specifically interacting are reported in Table S1. (E) Two-way hierarchical clustering of TACC3 and specific interactors CLTC, GTSE1, and PIK3C2A. Proteins significant binding in one of the pull-downs served as dataset for clustering (vertical direction). The color code represents the normalized log2 of ratios multiplied with the negative logarithmic p- values of the t test. Blue fields represent values close to 0, and the protein is therefore unlikely to be binding, whereas red fields represent highly specific binders in the distinct pull-down experiment. The first cluster represents a novel spindle-associated complex (red). The second cluster represents TACC3-specific interactors (green). The cluster marked in blue mainly consists of proteins associated with clathrin-coated vesicles. (F) Fluorescence microscopy showing live GFP fluorescence of TACC3, CLTC, and GTSE1 C-terminally tagged with GFP by the BAC TransgenOmics standard protocol. Both TACC3 interactors localize to the mitotic spindle. Bars, 10 µm.