Early Delivery of Misfolded PrP from ER to Lysosomes by Autophagy
Figure 9
Autophagy is selective for insoluble/aggregated PrP. (a) Representative Western blot of total lysates from COS-7 cells transiently expressing Wt- or Mut-PrP (3F4) after 16 h of incubation with 5 mM 3-MA. The corresponding graph displays the relative densitometry of PrP signal before (open bars) and after (filled bars) 3-MA treatment (*, student’s paired -test, ). Tubulin (TBLN) is a loading control. (b) Representative Western blots of supernatant (S) and pellet (P) fractions of cells transiently expressing Mut or Wt PrP after 16 h of vehicle (CTL) or 10 mM 3-MA, and the corresponding graph from 3 experiments each. The insoluble (pellet) and soluble (sup) fractions of Mut-PrP are presented as the densitometric signal of each fraction over the sum of the two fractions (sup + pellet) ± SEM (*, 2-way ANOVA, Bonferroni posttest). (c) Cells transiently expressing Mut-PrP were treated for 6 h with 10 mM 3-MA or 20 μM rapamycin, harvested in lysis buffer, and digested with 5 μg/mL Proteinase-K (PK) for 15 min at 37°C. Densitometry quantified as the intensity of each fraction (i.e., total PrP (open bar) or PK-resistant PrP (filled bar)), relative to their respective untreated normalized control fractions (** ANOVA, Bonferroni post test, difference from CTL, experiments).
