FIG. 7.
Anchorage-independent Ras/dnp53 cells phosphorylate Rb in suspension and maintain Rb dependency for proliferation. (A) Antibody phospho-Rb (Ser-795) recognizes Rb in proliferating (b) but not in quiescent (a) rat Schwann cells. Cells from growing colonies in soft agar were removed and spun onto coverslips and immediately fixed. The cells were immunostained for either a cytoplasmic protein S100 (c) or phospho-Rb (d and e, which show examples of cells negative and positive for phospho-Rb staining, respectively). (B) BrdU incorporation of cells after transfection with a nonphosphorylatable form of Rb (12) or control vector. Four NSdnp53Ras clones are shown. Clones 6 and 7 are p16INK4A positive; clones 8 and 9 are p16INK4A negative. NSLT are Schwann cells expressing SV40 LT. Transfected cells were monitored by determining GFP expression. The error bars indicate the standard deviations of triplicate assays.