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. 2020 Sep 28;31(2):141–156. doi: 10.1038/s41422-020-00416-2

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© The Author(s) 2020

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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Fig. 6 — a NRK cells were transfected with NS or Sec13 siRNA. Cells were treated with Tg for 0 or 6 h and analyzed by western blot using an antibody against BiP. b NRK cells were transfected with NS or Sec13 siRNA, and treated with Tg for the indicated times. Total RNA was subsequently extracted for semiquantitative RT-PCR analysis of XBP1 mRNA species (Xbp1 S: spliced XBP1 mRNA band; Xbp1 U: unspliced XBP1 mRNA band). c Flag-ATF6-expressing NRK cells were transfected with NS or Sec13 siRNA. Cells were treated with Tg for 0 or 6 h and analyzed by western blot using an antibody against Flag. d NRK cells were transfected with NS or Sec13 siRNA. Cells were treated with Tg for the indicated times and analyzed by western blot using an antibody against phospho-PERK (Thr980) and phospho-eIF2α (Ser51). e RFP-Sec61β-expressing NRK cells transfected with GFP-PERK were treated with Tg for 0 or 6 h and then observed by confocal microscopy. Scale bar, 5 μm. f Immunostaining of endogenous PERK and RFP in RFP-Sec61β-expressing NRK cells treated with Tg for 6 h. Scale bar, 5 μm. Regions outlined with white dashed lines are magnified. g NRK cells treated with Tg for 0 or 6 h were homogenized and the lysates were subjected to 1000× g centrifugation to discard the nuclei. The supernatant was ultracentrifuged in OptiPrep density gradient medium. The distribution of PERK in the fractions was monitored by western blot. 1 is the top fraction. h In vitro protein kinase assays. Membranes were collected from Fraction 4 of NRK cells treated with Tg for 6 h as described in g and mixed with ATP and the peptides eIF2α p(45–56) or mutant eIF2α p(45–56, S51A). Kinase reactions were resolved on SDS-PAGE and visualized by immunoblotting using an antibody against phospho-eIF2α (Ser51).