Fig. 5. Bud6p and Bud6^1-565p sediment with taxol-stabilised microtubules. (A,B) In vitro MT-binding assays were carried out using whole cell extracts from bud6 cells expressing either HA₃-Bud6p (A) or HA₃-Bud6^1-565p (B). Extracts were incubated with taxol-stabilised microtubules (Taxol-MTs), free tubulin in the presence of nocodazole (Noco-Tub) or buffer (–) as a control. Following centrifugation through a 10% (A) or 20% (B) sucrose cushion, supernatant and pellet (MT) fractions were assayed by western blot analysis. (C) MT fractions obtained as in A were subsequently resuspended in one of the following: 8 mM ATP, 10 mM GTP, 0.5 M NaCl or 2 M urea and subjected to sedimentation through a second sucrose cushion. Pellet and supernatant fractions were analysed by western blotting. Only urea treatment decreased Bud6p association to MTs. (D) Bud6^1-565p sedimented in association with MTs from whole cell extracts of bni1^CT bnr1 cells.