Figure 4.

CP309 copurifies with γ-tubulin and the centrosome-complementing activity. (A) PEG precipitates CP309 and γ-tubulin together with the centrosome-complementing activity. Clarified embryo extracts were fractionated into supernatants and pellets at the indicated concentrations of PEG8000. The supernatant (sup) and pellet (ppt) were analyzed by Western blotting probing with antibodies against CP309 and γ-tubulin. For complementing assays, the pellet fractions were resuspended in buffer, clarified, and passed through desalting columns to remove PEG. The number of asters reconstituted using the pellet fractions was counted in 50 random microscopy fields. (B) Quality of the reconstituted microtubules asters. Microtubule asters are divided into strong, medium, and weak categories depending on the amount of microtubules nucleated (see Figure 5A). More than 80% of the microtubule asters reconstituted using PEG pellets belong to the strong or medium categories. (C) Sucrose gradient fractionation of the centrosome-complementing activity. The 3% PEG pellet fraction from above was further purified by 5–40% sucrose gradient sedimentation; see lane L for proteins loaded. Fractions were analyzed by SDS-PAGE and Coomassie Blue staining (top) or by Western blotting probing with antibodies against CP309 or γ-tubulin (middle two panels). The number of asters formed (bottom) was counted in 50 random microscopy fields. (D) Mono S column chromatography of centrosome-complementing activity. The active fractions from the sucrose gradient (fractions 11–13) were further purified on a Mono S column (HR 5/5), see lane L for proteins loaded. The eluted proteins were analyzed as in C. The peak of complementing activity (fraction 7) lies between the γ-tubulin peak (fraction 6) and CP309 peak (fraction 8/9).