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Figure S1. Characterization of anti-Dgt antibodies, codepletion of Dgt proteins, and Dgt localization. (A) Immunoblotting of whole S2 extracts by anti-Dgt3–6 polyclonal antibodies generated in this study. 90% reduction of the targeted Dgt was detected (red arrowheads). (B) Comparison of expression level of endogenous and HA-tagged Dgt3–6 proteins by immunoblotting of whole S2 cell extracts using polyclonal antibodies. Relative intensity of each band was described (right). (C) Protein level of Dgt proteins after RNAi of a subunit of the Dgt complex or γ-TuRC. Reduction of multiple Dgt proteins was detected after single Dgt RNAi, except that the Dgt2-HA (which was ectopically expressed using nonnative promoter) was insensitive to RNAi of other Dgts, and Dgt4 knockdown had little effect on the stability of Dgt5. (D) Immunofluorescence microscopy revealed spindle localization of endogenous Dgt4 in S2 cells (green). The localization was undetected after Dgt4 RNAi. Blue, DNA. (E) GFP-Dgt5 signal was clearly detected in the kMT-rich area of the metaphase spindle, whereas it was sharply reduced at the spindle equator, which is exclusively occupied by non-kMTs (interpolar MTs). (E, top) GFP-Dgt5 (green) and mCherry-tubulin (red) were simultaneously observed in living metaphase cells using a wide-field microscope with a 100×/1.4 NA objective lens. A representative cell image is displayed. MTs present on the area of chromosome (spindle equator) are non-kMTs, and GFP-Dgt5 was hardly detected on those MTs. (E, bottom) Signal intensities of GFP and mCherry were measured along a 20-pixel-wide line (blue), and relative intensity was plotted. GFP intensity was greatly reduced in the middle. (F) GFP-Dgt5 is localized on kMTs. 10 min of colcemid treatment destabilized unstable MTs but not kMTs in the spindle, and GFP-Dgt5 was clearly detected on those remaining MTs. Together with the result in E, we suggest that Dgt5 is preferentially localized on the kMTs during metaphase. (G) FRAP analysis of GFP-Dgt5 in early anaphase spindles. Cells in early anaphase (separating sister chromatids are indicated by green arrows) were selected (n = 6) and small regions (1.9 ± 0.6 µm2) were bleached (yellow circle). Relative GFP intensity was plotted with SD. Rapid recovery of fluorescence was observed. Bars, 5 µm.