Fig. 9. Effect of A2 suppression by RNAi on the expression of p53 and BRCA1. (A) Western blotting for p53 after treating Colo16 cells with pS, pS-Luc and pS-A2. Total protein isolated from cells collected 72 hours post-transfection was separated on SDS/12% polyacrylamide gel, transferred onto PVDF and probed with a monoclonal mouse antibody against p53. The band intensities of p53 and hnRNP A2 shown in the graph were normalized to that of GAPDH. The p53 data are the means of four replicates. (B) Western blot analysis of BRCA1 after treating Colo16 with pS, pS-Luc and pS-A2. Total proteins isolated from cells harvested 72 hours post-transfection were separated on SDS/5% polyacrylamide gel, transferred onto PVDF and probed with a monoclonal mouse anti-BRCA1 antibody. Levels of BRCA1 protein, averaged from three replicates were calculated as the ratio of BRCA1 to GAPDH protein. A significant difference (*P<0.05) in protein levels in the pS-A2 transformed cells was observed compared to levels in control cells. (C) Northern blot analysis was carried out to determine the amount of BRCA1 mRNA in Colo16 cells collected 72 hours after transfection with pS, pS-Luc or pS-A2. The results were quantified by densitometry and the relative amount of BRCA1 was calculated using ß-actin as a control. Results are the means±s.e.m. of four experiments. A significant difference (**P<0.05) in BRCA1 level was observed in pS-A2 transfected cells compared to levels in control cells.