Two point mutations located farther into the stem structure of the shRNA (9G9 and B18A1) also reduced its ability to induce IFN even though the base-pairing was perfectly maintained in these mutants. Finally, replacing the 9-nt hairpin loop with a 7-nt loop that had been previously shown to abolish shRNA-mediated RNAi (loop A mutant) (46) eliminated sh-B971's ability to induce IFN, suggesting the importance of RNA processing in the induction. To determine whether the inability of the mutant shRNAs to induce IFN was due to lower expression levels, we performed northern blotting analysis of the shRNA expression on the wild-type and two mutants. The mutants A1/G and Loop A were chosen because their final siRNA products have exactly the same sequence as that of the wild-type sh-B971 and can thus be detected with the same efficiency by the same probe. Although sh-A/G and sh-Loop A were clearly unable to activate IFN-b promoter ( Figure 4A ), they were both expressed at levels comparable to those of the wild-type sh-B971 product ( Figure 4B) . Interestingly, the final siRNA product of sh-Loop A was slightly smaller than those of sh-B971 and sh-A1/G, suggesting that cleavage did occur and perhaps occurred one or 2 nt into the stem to compensate for the shorter loop.