(a) Binding of RZZ to the indicated versions of Spindly constructs isolated from insect cells determined by Surface Plasmon Resonance (SPR). The response plotted on the y axis is normalized for the molecular weight of the analyte to yield the stoichiometry of binding. The experiment was repeated three times with similar results. See Supplementary Table 3 for source data. (b) Immunoblot showing GFP-Spindly versions in mitotic cells treated as indicated (Lon, Lonafarnib). The experiment was repeated twice with similar results. (c-f) Representative images (c,d) and quantification (e,f) of relative kinetochore intensities of the indicated GFP-Spindly variants in nocodazole-treated HeLa cells transfected with siRNA to Spindly. The graphs show the mean kinetochore intensity normalized to the values of SpindlyFL. Each dot represents one cell. The sample size in (e) is: FL (n= 54 cells), FL/C602A (n= 55 cells), ΔN (n= 56 cells), ΔN/C602A (n= 56 cells), Δ274 (n= 51 cells) Δ274/C602A (n= 55 cells), Δ287 (n= 55 cells), Δ287/C602A (n= 51 cells) pooled from two independent experiments. See Supplementary Table 3 for source data. The sample size in (f) is: FL (n= 63 cells), FL/C602A (n= 57 cells), ΔCCS (n= 61 cells), ΔCCS/C602A (n= 59 cells) pooled from two independent experiments. See Supplementary Table 3 for source data.