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. 2021 Apr 28;11:9188. doi: 10.1038/s41598-021-88610-z

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© The Author(s) 2021

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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PACT is not required for spontaneous activation of Ser6- and Ser97-Ala PKR mutants. PACT-PKR DKO cells and PKR-KO cells were transfected with plasmids expressing either wt PKR or indicated PKR mutants. After 48 h, PKR activation (phospho-Thr446) was analyzed by western blot (a) and stress granule formation was quantified in transfected cells using eIF3 detection by immunofluorescence (b). (a) Detection of phospho-Thr446 PKR (PKR-pT446), PKR and PACT by western blot. Note the similar activation of PKR in PACT-positive and PACT-deficient cells. β-actin was detected as a loading control. Acquisition was made with a CCD camera (Fusion Solo-S, Vilber) using the Bio1D software (version 15.08). (b) Graph (generated with Microsoft Excell 2011) showing the percentage of stress granule-positive cells counted among PKR⁺ cells, for PACT-PKR DKO and PKR-KO cell lines (mean ± SEM) (n = 4). 2-way ANOVA (Graphpad Prism 7) was used to compare mutant and wt PKR in each cell line.