Fig. 1.

Ectopic expression of CHO (Cg) Orc1 induced loss of cell adhesion under conditions where ectopic expression of CgOrc2 did not. (A) CHO cells were transfected with the amount of either pCgOrc1 (●) or pCgOrc2 (○) indicated and then incubated for 36 hours before determining the fraction of unattached cells. (B) CHO cells were transfected with 1 µg of either pCgOrc1 (●) or pCgOrc2 (○) and then incubated for the times indicated before determining the fraction of unattached cells. Data with pCI alone was subtracted from data for pCgOrc1 and pCgOrc2. Shaded areas indicate the conditions under which pCgOrc2 and pCI gave indistinguishable results. (C) CHO cells were transfected with 1 µg of either pFCgOrc1 (FOrc1) or pFCgOrc2 (FOrc2) for the times indicated in hours post-transfection (post-T). FOrc1 and FOrc2 were quantified in the total cell population (attached plus unattached) by immunoblotting using anti-FLAG antibody. A single band was detected that migrated as ~100 kDa for FOrc1 and ~65 kDa for FOrc2. (D) CHO cells, transfected as in panel A, were separated into those that lifted off the dish (unattached) and those that remained attached. Half of the unattached cells and 10⁴ attached cells were assayed for either FOrc1 or FOrc2. (E) CHO cells, transfected as in panel B, were separated into attached and unattached cells before assaying for FOrc1 and FOrc2 for the times indicated. In each experiment, attached and unattached cell proteins were fractionated in the same gel, transferred to the same immunoblot, and detected with the same anti-FLAG antibody to ensure accurate comparison.