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Fig. 7. Rgf3p localizes early in mitosis. (A) Rgf3p-Myc₁₃ cells (KGY4401) were grown to mid-log phase at 32°C in YE medium, and synchronized in early G2 phase by centrifugal elutriation. Samples were collected every 15 minutes and analyzed for the abundance of Rgf3p-Myc₁₃ by immunoblotting of denatured cell protein lysates. Blotting with anti-PSTAIRE to Cdc2p from the same immunoblot served as a loading control. The septation index and the percentage of binucleate (anaphase) cells were also measured at each time point. (B) The level of Rgf3p-Myc₁₃ was determined in log-phase wild type cells (KGY246) (lane 1), rgf3-myc₁₃ (KGY4401) (lane 2), rgf3-myc₁₃ ace2::kan^R (KGY5160) (lane 3) and rgf3-myc₁₃ overproducing Ace2p from the nmt1 promoter (lane 4) was determined by immunoblotting of denatured protein lysates. The abundance of Cdc2p determined by immunoblotting the same protein gel with anti-PSTAIRE served as a loading control. (C) rgf3-GFP cells (KGY4407) synchronized in G2 phase by lactose gradient centrifugation and followed throughout a cell cycle. The percentage of binucleate (anaphase) cells, septated cells, and cells with Rgf3p-GFP rings were determined every 20 minutes. (D and E) rgf3-GFP nda3-km311 cells (KGY5400) were arrested by incubation at 19°C for 6 hours and released at time 0 by shift to 32°C. Samples were fixed in ethanol every 5 minutes to determine the percentage of cells with Rgf3p-GFP rings and a septum (D). (E) A representative image of these cells at the arrest point. (F) rgf1-GFP cells (KGY4407) were synchronized in G2 phase by lactose gradient centrifugation and followed throughout a cell cycle. The percentage of binucleate (anaphase) cells, septated cells and cells with Rgf1p-GFP rings were determined every 20 minutes.

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