Figure S4.

Sgo1 central region does not significantly contribute to CPC binding in vitro. (A–C) SEC profiles (top) and corresponding representative SDS-PAGEs stained with Coomassie (bottom) for the analysis of CPC_ISB10–280 and Sgo1_130–280 interaction (A), CPC_ISB10–280 and Sgo1_{130–280 4A} interaction (B), and CPC_ISB10–280 and Sgo1_1–415 or Sgo1_{1–415 4A} interaction (C). For easy direct comparison, control SDS-PAGE and chromatogram corresponding to Sgo1_1–415 are shown in Figs. S2 A and S4 C. A Superdex S200 10/300 GL (Cytiva) column pre-equilibrated with 25 mM Hepes, pH 8, 150 mM NaCl, 5% glycerol, and 2 mM DTT was used for A and B and 25 mM Hepes, pH 8, 250 mM NaCl, 5% glycerol, and 2 mM DTT was used for C. For easy direct comparison, control SDS-PAGEs and chromatograms corresponding to CPC_{ISB 10–280} (marked with an asterisk) are shown in two different panels, A and B. (D and E) Isotherms for the analyses of CPC_{ISB 10–280} interaction with Sgo1_1–415 (D) and CPC_{ISB 10–280} interaction with Sgo1_{1–415 4A} (E). 40 μl of 100 μM CPC_{ISB 10–280} was injected into 200 μl of 10 μM Sgo1_1–415 or Sgo1_{1–415 4A}. The ITC experiments were performed with 16 × 2.5-μl injections (0.5 μl first injection), 180 s apart, at 20°C. Top panels show raw ITC data; bottom panels show integrated heat data corrected for heat of dilution and fitted to a standard 1:1 binding model (Malvern Instruments MicroCal Origin software, v1.3). (F) Representative immunoblot for the analysis of Sgo1 levels upon Sgo1 depletion using siRNA oligonucleotides. Quantification of Sgo1/Tubulin ratio using uncalibrated OD values (normalized Sgo1/Tubulin ratio for siRNA Sgo1 is 28.8 ± 6.6). Three independent experiments; mean ± SD; unpaired two-sided t test; ****, P ≤ 0.0001. (G) Representative immunoblot of Sgo1-GFP constructs (Sgo1-GFP, Sgo1_Nmut-GFP, Sgo1_4A-GFP, or Sgo1_Nmut/4A-GFP) showing comparable expression levels. DP, differential power; mAU, milli absorbance units. Source data are available for this figure: SourceData FS4.