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. 2016 Apr 20;7:11407. doi: 10.1038/ncomms11407

Figure 2. SKAP binds microtubules at the α-/β-tubulin interface and competes with the Ndc80 complex for microtubule binding.

Figure 2

(a) Sequence alignment of SKAP135–225 showing amino acid conservation between Homo sapiens, Mus musculus and Rattus norvegicus. Asterisks indicate amino acids identified in cross-linking analysis with microtubules. (b) Intermolecular (blue) and intramolecular (red) cross-links found between SKAP135–225, α-1B and β-2B tubulin in cross-linking reactions with 10 μM SKAP135–225 and 10 μM taxol-stabilized microtubules. (c) Cartoon representation of tubulin (PDB ID: 3RYC) showing residues involved in cross-linking with SKAP135–225. Respective residues are highlighted in stick representation. (d) Representative SDS–PAGE (top) and quantification (bottom) of microtubule co-sedimentation assays with 3 μM SKAP135–225 and 3 μM taxol-stabilized or subtilisin-treated microtubules (mean±s.e.m., n=3, t-test: ***P<0.005). M, molecular weight marker; P, pellet fraction; S, soluble fraction. (e) Representative SDS–PAGE of the pellet fraction (left) and quantification (right) of microtubule co-sedimentation competition assay with 1 μM taxol-stabilized microtubules and 1 μM Ndc80 complex in presence of 0–80 μM SKAP135–225 (mean±s.e.m., n=3). (f) Representative images (left) and quantification (right) of fluorescence microtubule flow cell assay with 100 nM taxol-stabilized, HiLyte-647 labelled microtubules, 35 nM GFP-tagged Ndc80 complex and 0–4 μM mCherry-SKAP135–225 (mean±s.e.m., n=3). Scale bar, 3 μm.