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Fig. 2. Cellular distribution of DmCdc6. (A) Characteristics of the Dmcdc6 antibody used for this study. Crude extracts of Drosophila S2 cells were loaded and analysed by western blotting with Dmcdc6 antibody. The sizes of molecular weight markers are indicated at the right of the figure. Text indicates the position of the Dmcdc6 and a band that is apparently unrelated (as determined by the use of affinity purified antibodies). Those bands marked with an asterisk are degradation products of Dmcdc6 that increase in intensity as S2 cell fractions are aged. (B) S2 cells in mid-log phase were fractionated and analysed by western blotting. 500,000 equivalent cells were loaded per lane. C, cytoplasmic extract; Chr, chromatin-bound extract; Np, nucleoplasmic extract; T, total cell extract. (C) FACS analysis of exponential phase S2 cells. (D) Proliferating S2 cells were treated with aphidicolin (as described in Materials and Methods) and fractionated as in Fig. 2A. The top panel shows the distribution of DmCdc6 in cellular fractions of untreated cells (Ctrl) and cells treated with aphidicolin (Aph). The bottom panel shows a Coomassie Blue-stained gel of the region of gel between 14 and 21 kDa to show that the distribution of histones is as would be expected for an efficient fractionation. (E) FACS profile of control and aphidicolin-treated cells on days 1 and 2 of the experiment. The fractionation above corresponds to the day 1 sample, although no significant difference was seen between day 1 and day 2. (F) Total cell extract from 500,000 cells exponentially growing (Exp.) or quiescent (Q.) were analysed for the presence of Cdc6 by western blot. (G) FACS profile of nondividing S2 cells prepared as described in Materials and Methods. (H) Proliferating S2 cells were treated with ecdysone as described in Materials and Methods, and the total amount of DmCdc6 in untreated (–) and treated (+) was analysed by western blotting. (I) FACS profile of untreated cells (left) compared with cells that had been treated with ecdysone as described in Materials and Methods (right).
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