Fig. 4. The N-terminal domain of pEg2 binds to centrosomes assembled in Xenopus egg extract from sperm heads. Centrosomes were assembled in vitro by incubating demembranated sperm heads in Xenopus egg CSF extract (Stearns and Kirschner, 1994) in the absence (A-H) or presence (I-P) of 400 ng/µl of the recombinant Nt-pEg2-(His)6 protein. The reaction was performed in the absence (A-D and I-L) or presence of the microtubule depolymerising drug nocodazole (20 µM final concentration) (E-H and M-P). (A,E,I,M) Hoechst-stained DNA. (C,G) The endogenous pEg2 protein probed with mouse 1C1 monoclonal antibody (diluted 1:50). (K,O) The recombinant Nt-pEg2-(His)6 protein probed with mouse 6E3 monoclonal antibody (diluted 1:50). (B,F,JN) Centrosome stained with -tubulin antibody (diluted 1:1000). Fluorescein-conjugated anti-rabbit antibodies (diluted 1:500) and Texas Red-conjugated anti-mouse antibodies (diluted 1:500) were used as secondary antibodies. (D,H,L,P) Overlay. Scale bar, 10 µm. The localisation of the proteins was observed by fluorescence microscopy (DMRXA Leica); the images were acquired using a black and white camera and treated with the Leica-Q-Fish program.