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Fig. 7. Fkh2p and Mbx1p are phosphorylated during M-phase. (A) Cell cycle-specific changes in Fkh2p and Mbx1p mobility. Cultures of fkh2-3HA cdc25-22 (JM 2253) and mbx1-13myc cdc25-22 (JM 2252) cells were synchronised by transient temperature arrest and upon release to the permissive temperature, samples taken every 5 minutes for western blot analysis. Blots were probed with anti-HA antibodies to detect Fkh2p, anti-myc antibodies to detect Mbx1p, and anti-PSTAIRE antibodies to detect Cdc2p as an invariant loading control. The synchrony of the culture was confirmed by measurement of the septation indices over 300 minutes. (B) Fkh2p is a phosphoprotein. Fkh2-3HAp was immuno-precipitated from lysates prepared from an exponentially growing culture of fkh2-3HA cells (JM 2257) using the anti-HA antibody. Sample A was untreated, and samples B and C were incubated with calf intestinal phosphatase, either with or without phosphatase inhibitors. Samples were separated by SDS-PAGE and analysed by western blot with anti-HA antibodies.
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