FIG. 7.

Inverse correlation between endogenous Skp2 and R100T proteins. (A) Depletion of Skp2 increases the level of endogenous R100T. Pancreatic cancer AsPC1 cells, which carry a natural R100T mutation in Smad4, were transfected with siRNA against Skp2 (siSkp2) or the irrelevant siLack against Drosophila Smurf. The expression levels of Smad4 and Skp2 were assessed by Western blotting with the antibodies indicated. (B) The level of endogenous R100T decreases as the level of Skp2 increases in S phase. AsPC1 cells were serum starved for 24 h to reach a synchronized G₀ phase and then stimulated with 20% fetal bovine serum for the indicated time. Cell extracts were subjected to Western blotting analysis with anti-Smad4 or anti-Skp2 antibodies. The percentage of cell cycle phases is shown.