Early Delivery of Misfolded PrP from ER to Lysosomes by Autophagy
Figure 7
Mut-PrP colocalizes with autophagosomes. Confocal immunofluorescence of HeLa cells stably expressing GFP-tagged Mut- (a, c) or Wt- (b, d) PrP before (−) and after (+) incubation for 6 h with 50 nM Baf A1. Cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 5 min, then stained with anti-LC3B antibody and DyLight 649-conjugated Affini-Pure goat anti-rabbit IgG, prior to inverted confocal fluorescence microscopy imaging using a Marianas Yokogawa type spinning disk (original magnification, x100). Li’s method (NIH Image J plugin) was applied to determine the degree of colocalization of PrP::GFP (green) and LC3B (red). Graphs display the intensity correlation quotient (ICQ), which ranges from −0.5 (no correlation of signals) to 0.5 (complete correlation), between LC3B and Wt- or Mut-PrP in the absence (e) or presence (f) of BafA1. (***). There were no differences in any values between those treated with BafA1 and control cells. Actual values are (e) BafA1(−) Wt-PrP = , Mut-PrP = , (f) BafA1(+) Wt-PrP = , Mut-PrP = , cells each.
