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. 2022 Aug 10;29(8):831–840. doi: 10.1038/s41594-022-00814-7

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© The Author(s) 2022

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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Extended Data Fig. 3 — (a) Flow cytometry gating strategy of PI positive CAD5 cells. (b) PNGase-F digestion of cell lysates induced a shift in both murine wild-type PrP^C and mPrP^2cys, indicating that both moieties had undergone N-linked glycosylation to a similar extent. Non-adjacent lanes were merged from the same gel. (c) CAD5 Prnp^-/- cells expressing mPrP^2cys did not show an upregulation of the unfolded protein response, suggesting that mPrP^2cys did not undergo pathological degradation. Values are given as percentage of empty control vector (p-eIF2α / eIF2α / actin). One datapoint per group corresponds to a different cell culture passage. Two-sided, unpaired t-test. (d) A POM2/POM3 sandwich ELISA of COCS transduced with empty control, mPrP^C, mPrP^2cys and buffer control shows robust mPrP^2cys expression in transduced COCS, albeit significantly less than wild-type mPrP^C. Slices were harvested at 28 days post-transduction. One datapoint corresponds to an independent, biological replicate of 6–9 pooled slices. Ordinary, one-way Anova with Šídák’s multiple comparisons test, *: adjusted p-value = 0.039 (e) Reduced levels of mNG in Prnp^-/- (ZH3) COCS expressing mPrP^2cys. mNG immunoreactivity values are divided by actin immunoreactivity and expressed as percentages of empty control. Slices were harvested at 28 days post-transduction. One datapoint corresponds to an independent, biological replicate of 6–9 pooled slices. Ordinary, one-way Anova with Šídák’s multiple comparisons test. Raw, uncropped blots can be found in the Source Data supplement. (f) Quantification of mNG and Calb1 fluorescence intensity from experiments shown in Fig. 3d-f. One datapoint corresponds to a biological replicate, e.g. one organotypic cultured slice. Unpaired, two-tailed t-test without adjustment for multiple testing. P-values are as follows: 31 dpt, 5.2x10¹⁰ vg*ml⁻¹, mNG: 0.001; 31 dpt, 5.2x10¹⁰ vg*ml⁻¹, Calb1: 0.0496; 15 dpt, 1.4x10¹¹ vg*ml⁻¹, mNG: 0.0065; 15 dpt, 1.4x10¹¹ vg*ml⁻¹, Calb1: 0.001. ***: p ≤ 0.001, **: p < 0.01, * p < 0.05.

Source data