Mitotic spindle localization interdependencies of CLTC, TACC3, and GTSE1 by RNAi and after aurora A inhibition. (A) Live imaging of mitotic cells expressing GFP-tagged CLTC, TACC3, or GTSE1 after RNAi (72 h). GTSE1 is mislocalized after CLTC or TACC3 RNAi, TACC3 is mislocalized after CLTC, but not GTSE1 RNAi, and CLTC is not mislocalized by either TACC3 or GTSE1 RNAi. Two images of representative cells are shown for each condition. (B) Live imaging of mitotic cells expressing GFP-tagged CLTC, TACC3, or GTSE1 after treatment with the aurora A kinase inhibitor MLN8054. Inhibition of aurora A activity mislocalizes GTSE1 but not CLTC from the mitotic spindle. Two images of representative cells are shown for each condition. CON, control. Bars, 10 µm.

Pull-downs of pericentrin splice isoforms. (A–C) Volcano plots representing results of the label-free pull-downs of GFP-tagged pericentrin^short and pericentrin^long. The logarithmic ratio of protein intensities in the pericentrin/HeLa (A and B) and pericentrin^long/pericentrin^short (C) pull-downs were plotted against negative logarithmic p-values of the t test performed from triplicates. (A and B) The hyperbolic curve separates specific pericentrin-interacting proteins marked in black (red dotted line) from background (blue dots). (C) Proteins binding specifically to either form of pericentrin are marked in black. The dotted line represents the ratio of pericentrin^long/pericentrin^short. (D) Plotted relative intensities of all peptides identified from pericentrin^short (blue) and pericentrin^long (red). The N-terminal part of the protein was identified in both pull-downs, whereas there is a stretch of ∼500 amino acids unique to the pericentrin^short form (green box). The C terminus was deleted in pericentrin^short, and therefore, peptides from this region were only identified in the pull-downs of pericentrin^long (brown box).

Fluorescence analysis of pericentrin^long and pericentrin^short cell lines. (A) Diagram of pericentrin^long and pericentrin^short BAC constructs. Pericentrin^short lacks a 29.5-kb region of genomic DNA present in pericentrin^long, including the PACT domain (red). The green and yellow box represents the GFP cassette. (B) Pericentrin^long and pericentrin^short show distinct cell cycle localizations. Still images from videos of GFP fluorescence are shown. (top) Pericentrin^long localizes to centrosomes throughout the cell cycle. (bottom) Pericentrin^short only shows centrosomal localization in mitosis. (C) Immunofluorescence showing pericentrin^short localization to centrosomes. Mitotic but not interphase centrosomes are stained by anti-GFP (pericentrin^short), whereas anti-pericentrin antibody labels all centrosomes. Arrows point to the location of interphase centrosomes. (bottom) Enlarged images of the above boxed regions are shown, containing two prophase/prometaphase centrosomes and one interphase centrosome. Cells are stained for α-tubulin, GFP (pericentrin^short), pericentrin, and DNA. Bars, 10 µm.

SILAC and label-free pull-downs of CDC23. (A) SILAC pull-down of CDC23 versus the untagged HeLa cell line. Annotated proteins were specific interaction partners of CDC23 with a p-value of ratio significance <0.001. APC core proteins are separated from APC adaptors (CDC20 and FZR1) by intensity. (B) Volcano plot representing results of the label-free pull-down of CDC23. The logarithmic ratio of protein intensities in the CDC23/HeLa pull-downs were plotted against negative logarithmic p-values of the t test performed from triplicates. A hyperbolic curve separates specific CDC23-interacting proteins marked in black (red dotted line) from background (blue dots). The known components of the APC (C10orf104/ANAPC16 only recently characterized in parallel studies), several known APC adaptors, and one uncharacterized protein, C11orf51, show a significant ratio in combination with high reproducibility (positive log2 ratios). (C) Localization patterns of GFP-tagged CDC23 and the new component C11orf51 in interphase. Bars, 10 µm.

QUBIC: a method for mapping protein–protein interactions by combination of BAC TransgeneOmics and quantitative MS. (A and B) Two optimized AP-MS approaches of QUBIC are shown using either SILAC (A) or label-free (B) protein quantitation. (A) In SILAC experiments, the WT cell line without a BAC transgene is cultured in a medium containing the C¹²N¹⁴ form of lysine, and the tagged cell line is cultured in a medium containing the C¹³N¹⁵ form of lysine. Separate pull-downs using magnetic beads coupled to anti-GFP antibody are performed, and elutes merged directly after elution by in-column digestion. Peptides are identified by high resolution LC-MS/MS and quantified by directly comparing relative intensities of the light and heavy forms of each peptide present in the mass spectrum. Specific interaction partners show high H/L ratios, whereas background binders have a ratio of 1. (B) In label-free experiments, tagged and control cells are cultured in normal media, and separate pull-downs are performed. Eluates are not mixed but analyzed separately by LC-MS/MS. Proteins are quantified with the label-free algorithm in MaxQuant software.

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.

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.

SILAC pull-downs of the TREX core components. (A and B) Results of THOC2 (A) and THOC6 (B) analysis are shown. The GFP-tagged protein, serving as bait, is indicated in the title. Annotated proteins marked by a black dot were more abundant in the pull-down of the tagged cell line, with P < 0.01 in both the forward and reverse experiments. Blue dots represent proteins that were not significant interaction partners. (top left) Fluorescence microscopy was performed on fixed samples of the indicated cell line with anti-GFP antibodies (green), α-tubulin antibodies (red), and DAPI (blue). (bottom right) Anti-GFP staining only is shown. (C) Two-way hierarchical clustering of specific TREX interactors. Proteins with a ratio >2 and P < 0.1 in the forward and reverse experiments of one of the pull-downs served as dataset for clustering (vertical direction). The color code represents the multiplied ratios of the forward and multiplied inverted ratios of the reverse experiment in log scale. Blue indicates proteins with a ratio <1 or no ratio, and red indicates proteins with extremely high ratios. The first cluster represents the TREX complex, and adaptor proteins are separated from the core by the tree. The T complex clusters are shown below the TREX. Furthermore, several proteins binding to all TREX components, but with a lower ratio (yellow), have been identified (TREX-associated proteins). Proteins identified with very low ratios in only one of the IPs (bottom of clustering) are likely to be contaminants. (D) Pull-downs of all forward and reverse experiments have been treated as a single experiment, and forward were plotted against reverse experiments. TREX core and T complex are clear outliers as well as all TREX adaptors identified by the clustering. DDX39, a known interactor of the TREX complex, also shows a significant ratio in the combined analysis (Fig. S1). Bars, 10 µm.