Early Delivery of Misfolded PrP from ER to Lysosomes by Autophagy
Figure 2
Mut-PrP in lysosomes. (a) Indirect immunofluorescence confocal microscopy of fixed HeLa cells after 16 h expression of Wt- or Mut-PrP, detected with anti-mouse-PrP D13 F(ab) antibody. Red (PrP) and green (LAMP-1) channels are displayed separately to the left of merged images. Yellow indicates colocalization. (b) Live cell fluorescence microscopy of HeLa cells transiently expressing Wt-PrP::GFP or Mut-PrP::GFP in the presence of 50 nM LysoTracker Red shows comparable results with indirect immunoflourescence. (c) Western blot of total lysates of COS-7 cells transiently expressing Wt- or Mut-PrP after 16 h incubation with vehicle (−) or 0.1 μM Bafilomycin A1 (Baf A1) added to the media. PrP is detected with the D13 antibody. Graphic representation of densitometric signal of Western blot from 3 experiments is displayed below. (d) as in (c), but following incubation in the absence (−) or presence of 100 μM Leupeptin (Leu) or 20 μM E64, probed with D13. α-tubulin (TBLN) as a loading control in (c) and (d). Molecular weight markers (kDa) on the left. Densitometric quantification of blots in (c) and (d) is shown in graphs as relative intensity compared to untreated condition. *, ** (Student’s paired -test, experiments each).
