MBP-N Mars binds and stabilizes microtubules in vitro.

<p>(A) Scheme of recombinant proteins used in this study. (B) MBP-N-Mars binds to MTs <i>in vitro</i>. 1 µg of purified MBP-N-Mars was incubated with taxol-stabilized MTs at different concentrations before ultracentrifugation through a glycerol cushion. (C) Plot of bound fraction of MBP-N-Mars against MT concentration. The calculated Kd value is given above the curve. (D) MBP-N-Mars stimulates the assembly of MTs. The indicated amounts of MBP protein, MBP-N-Mars and MBP-C-Mars were incubated with tubulin solution. Taxol was used as a positive control. (E) Quantification of the tubulin in the pellet after sedimentation of the samples according to the same procedure as in (D). (F) MT dilution assay in the absence or presence of MBP-N-Mars. Top: Coomassie Brilliant Blue staining of the pellet samples separated by SDS-PAGE. Bottom: Quantification of the Coomassie staining results by LI-COR ODYSSEY SA system. (G, H) Microtubule bundling assay. The same amount of MBP (G) and MBP-N-Mars (H) was incubated with tubulin solution in the presence of a low concentration of taxol. Tubulin structures were fixed by formaldehyde, stained with tubulin-FITC antibody and imaged by fluorescence microscopy. Scale bar is 5 µm.