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Figure S2. Transient expression of FP-Repo-Man in HeLa cells. (A) Endogenous and FP-tagged Repo-Man detected by Western blotting HeLa cell lysates. Lysates were prepared from nontransfected HeLa cells and from HeLa cells transiently expressing FP-Repo-Man, with 20 µg total protein separated by SDS-PAGE and transferred to nitrocellulose for Western blotting with anti-Repo-Man peptide antibodies. Arrows point to the endogenous and FP-Repo-Man bands, which run at approximately 116 and 145 kD, respectively. The arrowhead marks a 64-kD protein that is also detected by the antibody but which is not down-regulated by RNA interference knockdown of Repo-Man and is therefore most likely the result of antibody cross-reactivity. (B-I) Mitotic defects associated with overexpression of FP-Repo-Man. Upon transient overexpression of FP-Repo-Man in HeLa cells, there was a reduction in the number of mitotic cells observed. Of those found, many showed evidence of one of two types of mitotic defects. One defect observed was decondensed (solid arrows) and/or fragmented (dotted arrows) chromosomes evident in metaphase (B-E), whereas the other was lagging chromosomes and incomplete cytokinesis (arrowheads) evident in late anaphase (F) through telophase (G-I). Bars, 10 µm. (J-O) Variation in FP-Repo-Man turnover kinetics throughout the cell cycle. FRAP analyses were performed on HeLa cells transiently expressing FP-Repo-Man to compare turnover rates of the fusion protein at different stages of the cell cycle. The photobleached region is indicated in each panel as a dotted circle, and a plot of signal recovery over time for this region is shown above the cell image. SoftWoRx data analysis software was used to calculate the mobile fraction and t1/2 (half-time for recovery). The relatively slow turnover rate of the nucleoplasmic pool of FP-Repo-Man during interphase (J) is maintained in early prophase (K) but increases dramatically when the same cell progresses through to late prophase (L), suggesting that the protein is now diffuse. This rapid turnover rate persists into prometaphase (M), but the transition to metaphase is accompanied by the accumulation of a small amount of FP-Repo-Man on chromatin, and this pool of protein shows a similar turnover rate to that measured during interphase (N). The larger chromatin pool of FP-Repo-Man observed in anaphase and telophase also shows this slower turnover rate (O). Bars, 5 µm.