Varying RNA and POPG concentration does not lead to rPrP-res that can induce PrP^Sc formation in SN56 cells.

<p>(A) rPrP PMCA reactions seeded with rPrP-res and serially propagated in the presence of 30 µg/ml of RNA and 3 different concentrations of POPG lipid (POPG lanes) or in the presence of 4.4 µg/ml of POPG and 3 different concentrations of mouse liver RNA (RNA lanes) were digested with PK and assayed in duplicate by immunoblot. (B) SN56 cells inoculated with cell culture medium alone (Mock) or rPrP-res made in the presence of 30 µg/ml RNA plus 2.2 or 6.7 µg/ml of POPG lipid. (C) SN56 cells inoculated with cell culture medium alone (Mock) or rPrP-res made in the presence of 4.4 µg/ml POPG plus 15 or 45 µg/ml of RNA. (D) SN56 cells inoculated with rPrP-res (Passage 6). The concentration in µg/ml of the RNA and POPG used is indicated in the parentheses. Lanes labeled 1× indicate PMCA products generated using the original POPG and RNA concentrations of 4.4 µg/ml and 30 µg/ml, respectively. (E) SN56 cells inoculated with rPrP-res (Passage 10). Labeling is as in panel D. The first lane is a 1∶100 dilution of undigested PrP from SN56 cells. Based on a standard dilution curve of PrP^Sc derived from SN56 cells persistently infected with 22L (first 3–4 lanes in panels B-D), the limit of detection of the immunoblot for PrP is equal to ∼1–3% of the total cell equivalents loaded. For all panels, blots were developed using the anti-PrP mouse monoclonal antibody 6D11. Molecular mass markers (kDa) are indicated on the right of each panel. Irrelevant lanes have been cropped and some lanes have been rearranged for clarity, but each immunoblot panel derives from a single film exposure.