The Sld2-11D protein level is important to promote DNA replication.
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A, YST1698 (JET1-1 GALp-sld2-11D ORC6-FLAG) cells were grown in YPARaffinose medium (Asyn) and arrested in G1 phase with alpha factor, and then the culture was split into six portions. Different amounts of galactose were added to each portion, and samples were taken at the indicated times (0–6 hours after galactose addition). The DNA contents of the samples were analyzed by flow cytometry. B, DNA content at 0, 4 and 6 hours of different galactose amounts are compared by overlay. C, Whole cell extracts were prepared from the same samples from A and analyzed by western blotting. Sld2 proteins and Orc6-FLAG proteins were detected with anti-Sld2 and anti-FLAG antibodies, respectively. *: non-specific background band.