N-Glycans and Glycosylphosphatidylinositol-Anchor Act on Polarized Sorting of Mouse PrPC in Madin-Darby Canine Kidney Cells
Figure 2
Physiological membrane localization of PrPC glycomutants.
(A) Characterization of glycomutants (PrPCG1, PrPCG2, and PrPCG3) and PrPCWT for the study by Western blot analysis, using an antibody directed against the 3F4 epitope. Clones with similar amounts of overexpressed 3F4 tagged PrPC as assessed by densitometric analysis of Western blots were used for these analyses (see graph). Relative expression of various PrPC forms is shown in percentages of PrPCWT that was set to 100%. (B) Assessment of plasma membrane (non-permeabilized) and intracellular (permeabilized) localization of PrPC glycomutants by confocal microscopy shows presence of PrPC at the plasma membrane and intracellularly (scale bar is 10 µm). (C) Assessment of DRMs localization of PrPC glycomutants by Triton X-100 extraction at 4°C and sucrose density gradient centrifugation showing correct localization of PrPC glycomutants with flotillin-positive DRM containing fractions.