Figure 4.

G-clamp-modified S-ONs demonstrate sequence-specific inhibition of p27^kip1. Immunoblot analysis of PI3K and p27^kip1 in CV-1 cells transfected with 2.5 μg/ml GS3815 cytofectin complexed in the absence (lane 1) or presence of a range of concentrations of the G-clamp-modified S-ON, ON4, (lanes 2–5), or 2-nt-mismatch G-clamp-modified S-ONs (lanes 6–9). The sequence for the 2-nt G-clamp p27^kip1 mismatch S-ON is 5′-TGG CTC XCT TGC GCC, where the X indicates the position of the G-clamp substitution and italics denotes the mismatch sites. The G-clamp-modified p27^kip1 antisense S-ON inhibited p27^kip1 by 98.8% at 90 nM, 95.4% at 30 nM, 81.0% at 10 nM, and 26.7% at 3 nM when normalized to PI3K and compared with p27^kip1 protein levels in the control lane (lane 1, GS3815 cytofectin only). No effects on p27^kip1 levels were seen in extracts treated with the 2-nt mismatch S-ON. At 90, 30, 10, and 3 nM of the mismatch S-ON, p27^kip1 was expressed at 112, 107, 119, and 100%, respectively, of the control lane.