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. 2003 Jun 1;31(11):2705–2716. doi: 10.1093/nar/gkg393

Figure 2.

Figure 2

Figure 2

siRNA duplexes with a 3′ or 5′ overhang or without overhang (blunt) are equally potent in mediating gene silencing in HeLa cells. (A) Inhibition of PTEN mRNA expression in HeLa cells transfected with the indicated amounts of siRNA molecules. The sequences and different terminal structures of the siRNAs molecules are shown on the left. Mutations in the mismatch molecules are indicated by arrowheads. Samples were analysed in parallel for the level of PTEN mRNA expression 24 h after transfection by real time RT–PCR (Taqman) analysis. PTEN mRNA levels are shown relative to the mRNA levels of p110α, which served as internal reference. Each bar represents triplicate transfections (± SD). (B) Inhibition of PTEN protein expression by use of siRNAs with different terminal structures. The cells were harvested 48 h after transfection of the indicated siRNAs (30 nM) (lanes 2–7) or GeneBlocs (30 nM) (lanes 8 and 9). Cell extracts were separated by SDS–PAGE and analysed by immunoblotting using anti-p110α, anti-PTEN or anti-phospho-Akt antibody. The amount of p110α was used as a loading control and control cell extracts from untransfected HeLa cells (UT) were loaded in lane 1. (C) Inhibition of p110β mRNA expression in HeLa cells transfected with the indicated amounts of siRNA molecules. p110β mRNA levels are shown relative to the mRNA levels of p110α, which served as internal reference. The ratio of p110β/p110α mRNA of untransfected HeLa cells is shown at the bottom (UT). Each bar represents triplicate transfections (± SD).