FIGURE 4.
Knockdown of Srebp2 only interferes with PrPSc accumulation in the absence of exogenous cholesterol. 22L-infected N2a cells (clone 5) were transfected with siRNAs against Srebp2 and nonsilencing siRNA (ns) either in the absence (A) or presence of FCS (B) or in medium supplemented with BSA/cholesterol (BSA/chol) (C) as an external source of cholesterol. Cells were lysed 48 h post-transfection and analyzed for total PrP (PK−) and PrPSc contents following proteinase K treatment (PK+). For PK− samples, only one-third of the sample was loaded for presentation purposes. GAPDH levels were determined for the PK− samples. Successful knockdown of Srebp2 was determined by using ab28482. Antibody 4H11 was used for PrP detection. Additional bands were excised for presentation purposes. Experiments were repeated four times, and results were normalized to GAPDH levels. PrPSc levels in Srebp2 siRNA-transfected cells were compared with PrPSc levels in nonsilencing siRNA-transfected cells (A–C, bottom panels). Significant changes are indicated by asterisks (**, p ≤ 0.005; paired t test). −/− denotes no significant differences.