Different localization of PrP in non-polarized and polarized MDCK cells in 2D and 3D, revealed by using different antibodies.

<p><b>(A)</b> Schematic representation of mouse PrP α-cleavage site and the recognition sites of antibodies used in this paper. The C-terminal part of PrP (orange) is membrane-attached via GPI-anchor; it has 2 independently occupied glycosylation sites (orange circles). Proteolytic α-cleavage occurs at the position 109*110. After the cleavage C-terminal fragment (C-ter) stays on the plasma membrane. C-ter as well as PrP full-length (FL) is recognized by SHA31 antibody (orange). The N-terminal part (blue), becomes a soluble N-terminal fragment (N-ter) upon cleavage. N-ter and PrP FL are recognized by SAF32 antibody (blue). <b>(B), (C)</b> and <b>(D)</b> Immunofluorescence images of MDCK cells stably expressing PrP (MDCK PrPwt cells). Non-polarized cells—plated for 24 hours on Matek (B), polarized MDCK -5 days on Transwell filters (C) 3D polarized cyst- 5 days in Matrigel^™ (D) were fixed and immunostained for PrP using SAF32 antibody (red) and SHA31 antibody (green) and nuclei are stained with DAPI (blue). Scale bars 10 μm. In <b>(C)</b> serial confocal sections of 0,3 μm were collected from the top to the bottom of the cell monolayer. <b>(E)</b> Pearson’s R values revealing colocalization of SAF32 and SHA31 signal in non-polarized, 2D and 3D polarized states. (**p ≤ 0.01, Mann-Whitney test) <b>(F)</b> Quantification of apical vs. basolateral distribution of SAF32 and SHA31 in 2D polarized MDCK cells (left panel) and in 3D polarized MDCK cysts (right panel). These experiments were performed 3 independent times (20 cells quantified per experiment) and a total of 60 cells were used for the final quantification. (** p ≤ 0.01, paired t-test).