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Fig. 10. Effect of A2 suppression by RNAi on the expression of p21. (A) Gene expression of p21 in Colo16 cells was determined by northern blotting using cells harvested 72 hours after transfection to analyse the effect of A2 suppression on p21. A densitometric analysis was performed to calculate the relative abundance of p21 mRNA. Results are the means±s.e.m. of three replicates. A significant difference (*P<0.01) in p21 mRNA level was observed in pS-A2 transfected cells compared to levels in control cells. (B) Total RNAs isolated from cells collected 72 hours after transfection were reverse-transcribed, and the abundance of hnRNP A2 and p21 mRNAs were determined by real-time PCR. The relative abundance, the mean (±s.e.m.) of three replicates, was calculated as the ratio between A2 or p21 mRNA and ß-actin. A significant difference (**P<0.05) in relative abundance of p21 mRNA was observed in pS-A2 transfected cells compared with levels in control cells. (C) Western blot analysis of p21 after suppressing hnRNP A2. Total proteins isolated from cells harvested 72 hours post-transfection were separated on SDS/12% polyacrylamide gel, transferred onto PVDF and probed with a monoclonal mouse anti-p21 antibody. The mean levels (±s.e.m.) of p21 shown in the graph were taken from duplicate experiments.
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