Figure 3

Mammalian cell gene silencing assay. Mouse BalbC 3T3 cells in a 24-well tissue culture plate format were transfected with equal amounts (500 ng each) of expression vectors for EGFP (pdsEGFP) and a mouse caspase 9/EGFP fusion (pmC9-dsEGFP) in the presence of the indicated concentrations (nM) of siRNAs. The fluorescent image (top) shows EGFP/c9EGFP expression in transfected cells. siRNAs used are as follows: Dc9si (gel purified Dicer-generated caspase 9); DGsi (gel purified Dicer-generated GAPDH); Esi (synthetic EGFP siRNA). DGsi/DGsi¹ and Dc9si/Dc9si¹ refer to two different siRNA preparations. Silencing was assessed at 16 hours post-transfection by Western blotting using EGFP antibody and 15% of the protein extract from a single well of the tissue culture plate (as described in materials and methods).