Early Delivery of Misfolded PrP from ER to Lysosomes by Autophagy
Figure 5
Mut-PrP expression activates autophagy. (a) Western blot of total lysates of N2a cells stably expressing Wt- or Mut-PrP engineered with the 3F4 epitope and probed for Atg12 (free and conjugated with Atg5), Beclin-1, phosphorylated mTOR, and LAMP-1. Loading was normalized for total protein. Tubulin (TBLN) is presented as a loading control. (b) Western blots of total lysates prepared from cells in (a) that were treated for 6 h with vehicle or 50 nM Bafilomycin A1 to limit LC3 degradation and probed for PrP with 3F4 mAb, LC3, and tubulin. PrP was detected by 3F4 mAb. The LC3II/LC3I ratio was measured by densitometry of each fraction, using Image One (BioRad) software from ECL developed Western blots, and displayed in the graph below (**, student’s -test, experiments). (c) Representative blots of total (T), supernatant (S), and pellet (P) fractions of cells in (a), following lysis and separation by centrifugation at 100,000 ×g for 1 h, were probed for PrP and LC3. LC3II/LC3I ratios for each fraction are presented in the graph below (*, student’s -test, ).
