Fig. 2. Changes of the Dd-EF2H protein during progression of cell cycle and starvation of synchronized cells. (A) Growth-phase Ax-2 cells synchronized by the temperature-shift method (Maeda, 1986) were withdrawn at the indicated cell-cycle phases. Exponentially growing Ax-2 cells (1-2x10⁶ cells/ml) at 22.0°C, with a doubling time of about 7.5 hours, were shifted to 9.5°C, shaken for 14.5 hours and then reshifted to 22.0°C. Under these conditions, the cell number doubled within about 2 hours after a lag phase of about 1 hour. Tt cells, t hours after the shift-up from 9.5°C to 22.0°C, were harvested for western blot analysis. In another experiment, T7 cells, 7 hours after the shift-up, were harvested just before the PS-point, starved by washing twice in 20 mM Na/K-phosphate buffer (pH 6.5), and shaken at 1x10⁷ cells/ml for 2 hours at 150 rpm. This yielded T7+2 cells, i.e. newly differentiating cells from the PS-point. T1+2 and T3+2 cells were also prepared by starving T1 and T3 cells for 2 hours in the buffer, as starved but not differentiated cells. The cell pellets were dissolved in 9 volumes of SDS-sample buffer, and the samples derived from the same number of cells (5x10⁴ cells) were applied to SDS-PAGE (10% gel), followed by transfer to PVDF membranes and western blotting. In another experiment, synchronized T1, T3 and T7 cells were starved in BSS for 2 hours to obtain T1+2, T3+2 and T7+2 cells, respectively. Their western blot analyses were carried out as described above. (B) A schematic representation of the cell-cycle of an Ax-2 cell. The checkpoint (PS-point) of growth/differentiation is interposed just after T7.

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