Bivalent Ab1 inhibits cell-cycle progression at G0/G1 phase. (A) Stable and control CHO cell lines (CD148-CHO, control-CHO) were prepared as described in “Materials and methods.” Cells were plated, serum-depleted for 48 hours, and pretreated for 60 minutes with serum-free medium containing Ab1, Ab1.Fab, and control IgG1 (67 nM). Then, the medium was replaced to growth medium containing 67 nM of each agent (day 0). Cell number was evaluated at days 1, 3, and 5. An upper panel displays anti-HA immunoblot on cell lysates (25 μg protein) prepared from the CHO lines. (B) The CHO cells, CD148-CHO and control-CHO, were plated in 100-mm dishes (1 × 105 cells/dish), synchronized by 72-hour serum depletion, and pretreated for 60 minutes with serum-free medium containing Ab1 or Ab1.Fab (67 nM), and the medium was replaced with growth medium supplemented with Ab1 or Ab1.Fab (67 nM) (time 0 hours). Cell-cycle progression was analyzed by FACS as described in “Materials and methods.” The blue indicates S phase; red, G0/G1 and G2/M phases. Bivalent Ab1 inhibits S-phase entry in CD148-CHO cells, while Ab1.Fab does not.
