Figure S1.

Additional information related to Figs. 1, 2, and 3. (A) Immunofluorescence images of EGFP-Mps1, Mps1^T490Ph, and Megator localization pattern in interphase control S2 cells and interphase S2 cells expressing EGFP-Mps1^WT, EGFP-Mps1^WT-NLS, or EGFP-Mps1^KD-NLS. To prevent nuclear export, cultured cells were treated with 10 µM Leptomycin B for 3 h. Graphs represent the intensity profiles of GFP-Mps1, Mps1^T490Ph, and Megator signal along the dotted lines. (B) Pull-downs of the indicated recombinant MBP-Megator fragments by bead-immobilized 6xHis-Mad1^1–493 or 6xHis-BubR1^359–696 (negative control). Beads (B) and flow-through (FT) were blotted for the indicated proteins. (C) Pull-downs of recombinant purified MBP-Megator^{1,187–1,655/T2D}, MBP-Megator^{1,187–1,655/T3D}, MBP-Megator^{1,187–1,655/T4D} and MBP-Megator^{1,187–1,655/T4A} by bead-immobilized 6xHis-Mad1^1–493 or 6xHis-BubR1^1–358 (negative control). B and FT were blotted for the indicated proteins. (D) Quantification of MBP-Megator binding to 6xHis-Mad1^1–493 from pull-downs in C. The graph represents the ratio between the signal intensities of MBP-Megator and 6xHis-Mad1^1–493. The value obtained for MBP-Megator^{1,187–1,655/T4A} was set to 1. (E and F) Representative immunofluorescence images (E) and corresponding quantifications (F) of Mad1 at the NE of interphase S2 cells depleted of endogenous Megator and expressing the indicated Megator-EGFP transgenes. Mad1 fluorescence intensities were determined relative to Nup107 signal (n ≥ 24 cells). In F, data are presented as mean ± SD. ***, P < 0.001 (Student’s t test). Scale bars, 5 µm (inset, 0.5 µm).