Fig. 3. The N-terminal domain of CAP350 binds microtubules directly. (A,B) The fragment (403-725) produced in bacteria as a GST fusion protein and purified using the GST tag, as well as GST, were incubated for 1 hour with preformed MTs. The MTs were then centrifuged on a glycerol cushion and the proteins from the supernatants (S) and pellets (P) were analysed by SDS-PAGE. (A) Coomassie Blue staining. The arrow indicates either purified GST fusion protein or GST. Asterisks indicate tubulin. (B) Western blotting using anti-GST antibody. (C) Double immunofluorescence of the microtubule-binding domain (403-725) mutated in the two NLS in fusion with the 6xMyc tag using anti-myc (left and green in merged image) or -tubulin antibody (middle and red in merged image). As expected, it colocalised with the microtubule network. Bar, 20 µm.