FIGURE 8.
Amcinonide reduced PrPC levels by increasing its rate of degradation. A, immunocytochemistry of N2a cells treated with 50 μm amcinonide for 3 days. Cells treated with DMSO were used as control. The negative control indicates DMSO-treated cells without the addition of D18 primary antibody. The green color corresponds to the PrPC (D18) labeling. The blue color locates the nuclei DAPI staining. B, dose-response analysis of the effect of amcinonide on PrPC expression levels. N2a cells were treated for 3 days with the indicated concentrations of amcinonide and PrPC levels were analyzed by Western immunoblotting. Actin staining (bottom) was performed on the same membrane used to detect PrPC expression. C, Prnp mRNA expression levels in N2a cells treated with 50 μm amcinonide for the indicated durations. All mRNAs levels were normalized with respect to actin controls. D, measurement of PrPC half-life after the addition of cycloheximide in cells treated with DMSO (top) or 20 μm amcinonide (bottom). Blots were probed with D18 anti-PrP antibody. For Western blots, apparent molecular mass markers are indicated in kilodaltons.