Fig. 4. Depletion of Top3 leads to phosphorylation of Chk1. (A) Growth curves of exponentially growing cultures of the top3-P41 strain in the presence () or absence () of thiamine at 36°C. (B) Cell and nuclear morphologies of samples from A were determined by fluorescence microscopy of DAPI-stained cells 12 hours after the addition of thiamine. Bar, 10 µm. (C) Whole-cell protein extracts were prepared by trichloroacetic acid precipitation following glass bead disruption. The extracts were separated by SDS-PAGE and subjected to immunoblotting using anti-HA (upper panel) or anti-Cdc2 (loading control) antibodies as indicated.