FIG. 6.

BRCA1-dependent ubiquitination activity inhibits aster formation in vitro. (A) Centrosomes purified from Hs578T cells were preincubated with ubiquitination factors comprising E1, E2, and ubiquitin (Ub) and with 30 nM BRCA1/BARD1 (B/B) as indicated and were then allowed to form asters in the presence of Xenopus extract. The centrosomes were then spun onto glass coverslips and visualized by double staining with antibodies against α-tubulin (red) and γ-tubulin (green). Representative fields are shown containing asters formed after treatments as labeled in the bottom right corner of each panel. Centrosomes were preincubated without ubiquitination factors or BRCA1/BARD1 (top left), with ubiquitination factors (top right), with both ubiquitination factors and BRCA1/BARD1 (bottom left), or with BRCA1/BARD1 but without ubiquitination factors (bottom right). Bar, 10 μm. (B) The mean of the product of fluorescence intensity and area (Mt content), expressed in arbitrary fluorescence units, was calculated for 40 asters from each condition explained above for panel A in two independent experiments. The average values were normalized with respect to the control reaction, which had no BRCA1/BARD1, E1, E2, or ubiquitin added (set at 100%), and values are shown as percentages with the standard errors of the mean. (C) Centrosomes purified from HeLa S3 cells were preincubated with ubiquitination factors as for panel A, except that in each reaction a single component was omitted. The MT content of asters was calculated as described for panel B, and the average values normalized to a control reaction (set at 100%) are shown with standard errors of the means.