FIG. 9.

Cell cycle arrest after overexpression of Skp2 in mammalian cells. (A) HtTA1 cells were transiently transfected with the same amount of vector pcDNA3.1(−) (lane 1) or Skp2 in pcDNA3.1(−) (lane 2). Cell extracts were prepared and subjected to SDS-PAGE (17.5% gel). Proteins were transferred to a membrane and immunoblotted with antibodies against Skp2 and Skp1. Positions of the endogenous Skp1 and transfected Skp2 are shown on the right; positions of molecular size standards (in kilodaltons) are shown on the left. (B) HtTA1 cells were transiently transfected with the same amount of vector pUHD-P1 (lane 1) or FLAG-Skp1 in pUHD-P1 (F-Skp1; lane 2). Cell extracts were prepared and subjected to SDS-PAGE (17.5% gel). Proteins were transferred to a membrane and immunoblotted with antibodies against Skp2 (top) and FLAG-tag (bottom). Positions of the endogenous Skp2 and transfected FLAG-tagged Skp1 are shown on the right. (C) HtTA1 cells were cotransfected with Skp2 in pcDNA3.1(−) construct and a plasmid expressing CD20. Immediately after harvest, the cells were incubated with a fluorescein isothiocyanate-conjugated anti-CD20 monoclonal antibody, fixed, and stained with propidium iodide. DNA content of the transfected cells (upper portion of the cell population diagram) and nontransfected cells (lower portion of the cell population diagram) was analyzed by FACS. (D) HtTA1 cells were cotransfected with FLAG-tagged Skp1 in pUHD-P1 and a CD20-expressing plasmid as for panel C. FACS analyses of the transfected and nontransfected cells are shown.