Fig. 1. Dose-response effect of siRNA duplexes on PrP-sen expression and PrP-res accumulation in infected N2aS12sc⁺. (A) Immunoblot of a representative experiment performed with transfected N2aS12sc⁺ cells in the absence (0) or in the presence of increasing concentrations of Prnp gene-specific siRNA or of scrambled siRNA duplexes. One-tenth of the post-nuclear cell lysates were directly mixed with the same volume of 2x denaturing loading buffer. PrP-sen was first detected with the SAF83 mouse monoclonal antibody. The same blot was then incubated in the presence of rabbit polyclonal antibodies raised against ERK proteins (40-42 kDa) as a control of protein loading. (B) 90% of the lysates of untransfected or siRNAs-transfected N2aS12sc⁺ cells were PK-digested as described under Materials and Methods and loaded onto a 12% polyacrylamide gel. Partially PK-digested PrP-res was assayed with SAF83 monoclonal antibody. Molecular mass markers are indicated on the left in kilodaltons (kDa). (C) Densitometry analysis performed on blots developed and exposed as described above were done with `National Institutes of Health' IMAGE software. The results of four independent experiments are shown and expressed as a percentage of control levels ± s.e.m. (bars).