Fig. 4.

CCDC74A/B are microtubule-binding proteins. a Microtubule (MT) co-sedimentation assays in vitro. CCDC74B (0.2 μM) was expressed in E. coli then purified and incubated with or without taxol-stabilized microtubules in BRB80 buffer. After centrifugation, supernatants (S) and pellets (P) were separated and stained with Coomassie blue (CBB). b Schematic of GST-tagged CCDC74A/B full-length and their mutants, illustrating microtubule-binding activity of CCDC74B (+, positive; −, negative). c–e Western blot analysis of microtubule co-sedimentation assays in vitro. GST or GST-tagged full-length (1-314 aa) CCDC74B or the mutants in b were expressed in E. coli, purified, and incubated with taxol-stabilized microtubules in BRB80 buffer (+, present; −, not present). Supernatants and pellets were separated by centrifugation then stained with CBB. f–h GST or GST-tagged full-length CCDC74B or the indicated mutants (0.1 μM) bound to glutathione-sepharose 4B beads were incubated with taxol-stabilized microtubules in BRB80 buffer at room temperature in vitro. The bead-bound proteins were analyzed by Western blotting with anti-tubulin antibody and by CBB staining. GST served as a negative control