FIG. 6.
Functional assay of pRNA/siRNA chimera targeting proapoptotic factor BAD. (A) pRNA/siRNA(BAD) and control siRNAs (10 nM) were introduced into pro-B cells by electroporation, combined with a transfection reagent on day 1. Cells were washed to remove IL-3 on day 2 and assayed for viability on day 3. (B) BAD protein levels were compared in cells transfected with chimeric siRNA(BAD) or two mutant controls containing different mutations within siRNA sequences. Control cells were treated with pRNA alone. Cell lysates were prepared (Khaled et al., 2001) on day 3 and proteins were separated by 12% SDS–PAGE followed by Western blot with BAD antibody (Cell Signaling Technology, Beverly, MA). Numbers below the panel indicate the remaining BAD level, expressed as a percentage compared with control cells. (C) Pro-B cells transfected with pRNA/siRNA(survivin) or mutant were grown in complete medium containing IL-3 or deprived of IL-3. The impact of RNA on cell morphology was observed by microscopy.