Figure 8: Electrostatic interactions mediate CGN binding to microtubule CTTs.

(a) Microtubule binding assay showing amount of pelleted GST-CGN(1–406) and GST-CGN(1015–1203) when incubated with (+) and without (−) taxol-stabilized microtubules (tubulin). Anti-GST western blot (top gel), and Coomassie stained SDS–PAGE gel (bottom gel) are shown. (b) Diagram depicting positioning of α- and β-tubulin CTTs within polymerized microtubule. (c) Amino acid sequence alignment comparing CTTs of human and yeast β-tubulins. (d) Microtubule binding assay comparing amount of CGN(1–406) that pellets with wild-type tubulin and subtilisin-digested tubulin. Coomassie stained SDS–PAGE gel is shown. (e) Microtubule binding assay comparing CGN(1–406) pelleting with wild-type yeast tubulin and mutant yeast tubulin containing no β-CTT. Anti-tubulin and anti-CGN western blot is shown. (f) Amino acid sequence alignment comparing CGN basic patch in different vertebrate species. Boxes mark the sites mutated to alanine for binding analysis shown in (g). (g) Quantification comparing microtubule binding of wild-type CGN(1–406), CGN-M1 (R36/37A) and CGN-M2 (R40/41A). Data shown are the means and s.d. derived from four independent binding experiments.