JCS035360 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. Eg5 interacts with Nercc1 and Nek6 through its C-terminal tail in the yeast two-hybrid system. The ability of Eg5 head (Eg5 1-361), stalk (Eg5 362-761) and tail (Eg5 762-1057) domains to interact with Nek6 or Nercc1 was assessed using two-hybrid by histidine and adenine prototrophy plus expression of α-galactosidase activity (left plate in both pictures). Fusion-protein expression was verified by immunoblot (upper panel). AD, BD: Gal4 activation and binding domains. The interaction between p53 and SV40 is a positive control.

• Supplemental Figure S2 -

  Fig. S2. Characterization of anti-HsEg5Ser1033-P and anti-XlEg5Ser1046-P antibodies. Characterization of anti-HsEg5Ser1033-P antibodies: (A) Recombinant GST-Eg5 wild type, purified by glutathione affinity from extracts of HEK293T cells, was either left untreated (left lane) or incubated in phosphorylation buffer containing ATP/Mg²⁺ (middle and right lanes) and either NIgG (−) or FLAG-Nek6 (+) immunoprecipitates. After SDS-PAGE, immunoblotting with affinity purified anti-Eg5Ser1033P antibody (top panel) was carried out; a Coomassie blue stain of the GST-fusion proteins is shown (bottom panel). (B) Recombinant GST-Eg5 wild type or GST-Eg5Ser1033Ala, purified as in A, were incubated with FLAG-Nek6 in the presence of ATP/Mg²⁺. Top panel: immunoblot with affinity purified anti-Eg5Ser1033P antibody; bottom panel: Coomassie blue stain of the GST-fusion proteins. (C) Bacterial-expressed forms of the Eg5 C-terminal tail fused to Maltose binding protein (MalBP), MalBP-762-1057 and MalBP-Eg5762-1057/Ser1033Ala were incubated with ATP/Mg²⁺ and either NIgG (−) or FLAG-Nek6 (+) immunoprecipitates. Immunoblot with affinity purified anti-Eg5Ser1033-P antibody as in A. Characterization of anti-XlEg5Ser1046-P antibodies: (D) Bacterial recombinant MalBP-XlEg5773-1067 was incubated with ATP/Mg²⁺ and either NIgG (−) or FLAG-Nek6 (+) immunoprecipitates. XlEg5Ser1046 phosphorylation was visualized by immunoblot with anti-XlEg5Ser1046-P antibody (top panel); MalBP-fusion proteins were stained with Coomassie blue (bottom panel). (E) XL177 cell extracts (50 µg of total protein) were diluted 1:5 in lambda protein phosphatase buffer (New England Biolabs; final concentration 1×) and incubated for 30 minutes at 30°C minus or plus lambda protein phosphatase (800 U). XlEg5Ser1046-P and total XlEg5 were visualized by immunoblot (top and lower panels, respectively).

• Supplemental Figure S3 -

  Fig. S3. Microtubule binding affinity of Eg5 wt, T926A, S1033A and S1033D. (A) Loading quantification of recombinant Eg5. His₆-Eg5 wild type (wt), Thr926Ala (TA), Ser1033Ala (SA) and Ser1033Asp (SD) were purified from Sf9 cells and equalized for protein content. (B) Microtubule binding assay. 5.7 µg of recombinant Eg5 was incubated with polymerized microtubules and pelleted by centrifugation. Representative aliquots of the supernatants (S) and pellets (P) was analyzed by immunoblot of Eg5 (upper panel), whereas tubulin abundance was determined by Coomassie blue stain (lower).

• Supplemental Figure S4 -

  Fig. S4. Subcellular localization of different recombinant Eg5 forms in cells arrested in mitosis with monopolar spindles by endogenous Eg5 depletion. HeLa cells co-transfected with Eg5 siRNA and the indicated RNAi-resistant myc-tagged Eg5 forms (as in Fig. 5), were fixed with methanol and immunostained with anti-myc to visualize Eg5. Note the accumulation of S1033A and S1033D (but not T926A) forms at the center of the cell over the collapsed spindle.