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. 2017 Aug 29;7:9600. doi: 10.1038/s41598-017-08821-1

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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β-cleavage of PrP^C modulates intracellular iron and is unique to polarized RPE cells. (a) Deglycosylated lysates from vector and PrP^C-expressing M17 and RPE cells and human brain were probed with PrP-specific antibodies 3F4 and anti-C to estimate the relative abundance of full-length (FL), α-cleaved (C1), and β-cleaved (C2) forms in each sample. Probing with 3F4 that reacts with FL and C2 (Fig. 4a) reveals undetectable levels of C2 in M17 cells even after over-expressing PrP^C (lanes 1 & 2). RPE cells, on the other hand, show significantly more C2 in non-polarized cells (lanes 3 & 4), and almost complete cleavage of FL to the C2 form following polarization (lanes 5 & 6). The brain sample shows minimal levels of C2 as in M17 cells (lane 7). Probing with anti-C antibody that reacts with FL, C1, and C2 (Fig. 4a) shows FL and C1 in M17 and brain lysates (lanes 8, 9, 14), and mainly C2 in RPE samples (lanes 10–13). Re-probing for TfR shows down-regulation following polarization of RPE cells (lanes 3 & 4 vs. 5 & 6). Re-probing for DMT-1, on the other hand, shows up-regulation in PrP^C-expressing non-polarized RPE cells (lanes 10 & 11), and down-regulation following polarization (lanes 10 & 11 vs. 12 & 13). (b) Densitometric analysis of protein bands after normalization with β-actin shows downregulation of TfR following polarization of RPE cells to less than half, and upregulation of DMT-1 in PrP^C-expressing non-polarized cells by ~3-fold. DMT-1 expression is minimal in polarized RPE cells. Values are mean ± SEM of the indicated n. ns, not significant, *p < 0.05, **p < 0.01, ^##p < 0.01, ^###p < 0.001. (c) Exposure of non-polarized RPE cells to FAC increases the ratio of C2 vs. FL that is more evident following deglycosylation (lanes 5 & 7 vs. 6 & 8; d). Re-probing for ferritin shows a significant increase following FAC treatment as expected (lanes 2, 4, 6, 8).