(A) Endogenous SBDS co-pellets with microtubules from cell lysates. Increasing concentrations of purified, taxol-stabilized bovine microtubules were incubated with HeLa cell extract, layered over a glycerol cushion, and centrifuged to pellet the microtubules and associated proteins. The pellets were analyzed by western blot for tubulin and SBDS. (B) Recombinant SBDS co-sediments with microtubules. Left: Coomassie-stained gel loaded with standard molecular markers (lane 1) or 3 μg purified recombinant SBDS protein (lane 2). Upper right: The fraction of 500 nM SBDS remaining in the supernatant (s) or co-pelleting (p) with increasing amounts of taxol-stabilized microtubules after centrifugation through a glycerol cushion was monitored by SBDS immunoblotting. Lower right: The average percentage bound from 3 experiments. The 1-site binding isotherm and K_d = 7.6 ± 3.0 μM were obtained by best-fit nonlinear regression using Graphpad Prism software. Approximately 14% of purified SBDS was co-pelleted with microtubules. Error bars represent SEM. (C) SBDS stabilizes microtubules in vitro. Preformed fluorescence-associated microtubules were diluted into PBS buffer alone or into buffer containing either taxol or increasing concentrations of purified, bacterially expressed recombinant SBDS protein. SBDS concentrations of 1.74 μM, 3.5 μM, and 7 μM were assayed. Dilution-induced depolymerization was monitored by the loss of microtubule-stimulated fluorescence signal. Three independent experiments yielded similar results. A representative experiment is shown. As a negative control, purified bovine serum albumin protein had no effect on microtubule stability (Supplemental Figure 4).