FIG. 2.

Levels of Cdc18 and Orc RNAs and proteins in different stages of the cell cycle. (A) HeLa cells were synchronized in mitosis by nocodazole (50 ng/ml), and after shake off, the cells were replated in nocodazole-free medium. A Northern blot of RNA extracted from the cells at the indicated number of hours after release is shown. AS, asynchronous cells; hsorc1 and -2, hOrc1 and -2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (B) HeLa cells were either asynchronous (AS), blocked in S phase with hydroxyurea (HU), or blocked in mitosis with nocodazole (NOC). RNA, Northern blot of total RNA with the indicated probes; PROTEIN, immunoblots of protein extracts with preimmune serum (pre-imm) (for anti-Cdc18 antibody; the same result was obtained with the preimmune serum for anti-Orc1 antibody or rabbit serum against bacterially produced GST-Cdc18 protein (Cdc18) or the His₆-tagged C-terminal one-third of hOrc1 protein (hsorc1). (C) NIH 3T3 cells were released from serum starvation and harvested at indicated time-points (h) as the cells moved from G₀ through G₁ to S. Extracts were immunoblotted for the indicated proteins. Cyc A, cyclin A.