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.