Specificity controls for siRNA mediated c-Myb knockdown. (A) A flag-tagged c-Myb mutant (Myb^*) with a silent mutation in the siRNA target sequence was constructed by site-directed mutagenesis. Myb^* was inserted into pMSCV-MLL-ENL under control of the plasmid-borne phosphoglycerate kinase promoter and tested for expression by anti-flag immunoblot with wild-type c-Myb as control. When tested under identical conditions, Myb^* was significantly more resistant than wild-type Myb to the knockdown effect of anti-Myb shRNA (compare with Figure 3A). The constructs coding for MLL-ENL or MLL-ENL/Myb^* were cotransduced with shRNA expression constructs as indicated into hematopoietic precursors and tested for colony-forming ability after serial replating. A representative example of third-round colonies is shown. (B) The effect of shRNA-mediated c-Myb suppression on E2A-HLF-mediated transformation was tested in an experiment analogous to that in panel A.