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. 2019 Jan 23;9:410. doi: 10.1038/s41598-018-37207-0

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© The Author(s) 2019

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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Pharmacologic inhibition of PERK signaling disrupts ^FTTTR^A25T secretory proteostasis during Tg-induced ER stress. (A) Representative autoradiogram of [³⁵S]-labeled ^FTTTR^A25T in lysates and media collected from HEK293T cells pretreated for 16 h with thapsigargin (Tg; 500 nM) and/or ISRIB (200 nM). The protocol for the experiment is shown above. (B) Graph showing the total amount of [³⁵S]-labeled ^FTTTR^A25T at t = 0 h in HEK293T cells pretreated for 16 h with thapsigargin (Tg; 500 nM) and/or ISRIB (200 nM). Data are shown normalized to vehicle-treated cells. Error bars show SEM for n = 3 independent experiments. A representative autoradiogram is shown in (A). (C) Graph showing the fraction [³⁵S]-labeled ^FTTTR^A25T secreted at t = 4 h in HEK293T cells pretreated for 16 h with thapsigargin (Tg; 500 nM) and/or ISRIB (200 nM). Fraction secreted was calculated as described in Materials and Methods. Error bars show SEM for n = 3 independent experiments. A representative autoradiogram is shown in (A). (D) Graph showing the fraction [³⁵S]-labeled ^FTTTR^A25T remaining at t = 4 h in HEK293T cells pretreated for 16 h with thapsigargin (Tg; 500 nM) and/or ISRIB (200 nM). Fraction remaining was calculated as described in Materials and Methods. Error bars show SEM for n = 3 independent experiments. A representative autoradiogram is shown in (A). (E) Graph showing the fraction [³⁵S]-labeled ^FTTTR^A25T in lysates at t = 4 h in HEK293T cells pretreated for 16 h with thapsigargin (Tg; 500 nM) and/or ISRIB (200 nM). Fraction lysate was calculated as described in Materials and Methods. Error bars show SEM for n = 3 independent experiments. A representative autoradiogram is shown in (A). (F) Graph showing the recovery of BiP in anti-FLAG IPs from HEK293T cells expressing ^FTTTR^A25T and pretreated for 16 h with Tg (500 nM) and/or ISRIB (200 nM). Data are shown normalized to Tg-treated cells. Error bars show SEM for n = 3 independent experiments. A representative immunoblot is shown in Fig. S1F. *Indicates p < 0.05; ** indicates p < 0.01 for a paired two-tailed t-test.