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. 2021 Mar 16;12:1684. doi: 10.1038/s41467-021-21931-9

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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 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. 4 — a Conserved consensus sequence of Xbp1s-binding site (unfolded protein response element—UPRE) in the promoter region of STUB1. 129 bp from the transcriptional start site (arrow). b Luciferase (Luc) activity in human embryonic kidney 293 cells (HEK293) transfected with STUB1-luciferase reporter construct (STUB1-Luc) and transduced with the increasing multiplicity of infection of AdGFP or AdXbp1s (n = 6 biologically independent experiments). c Electrophoretic analysis of chromatin immunoprecipitation (ChIP) assay of STUB1 promoter in NRVMs transduced with AdGFP or AdXbp1s. ChIP was performed with either control mouse immunoglobulin G (IgG) or anti-Xbp1s antibody. PCR was conducted with primers spanning the UPRE site. Images are representative of 4 independent experiments. d Densitometric analysis of STUB1 ChIP band intensities in the different experimental groups (n = 4 biologically independent experiments). e mRNA level of STUB1 in NRVMs transfected with STUB1-specific small interfering RNA (siSTUB1) or scrambled siRNA control and transduced with AdGFP or AdXbp1s (n = 4 biologically independent experiments). f Representative immunoblot images of Xbp1s, STUB1 and GAPDH proteins from NRVMs transfected with siSTUB1 or scrambled siRNA control and transduced with increasing multiplicity of infection of AdGFP or AdXbp1s. Images are representative of five independent experiments. g Densitometric analysis of STUB1 protein band in the different experimental groups (n = 5 biologically independent experiments). h Representative immunoblot images of Xbp1s, STUB1, and GAPDH proteins from LV of WT and Xbp1s TG mice. High and low exposure (high/low exp). Images are representative of six hearts/group. i Bar graphs depicting STUB1 protein band intensities in WT and Xbp1s TG LV samples (n = 6/group). j mRNA level of XBP1s and STUB1 in LV of CTR and HFpEF hearts (n = 4 for Xbp1s CTR group; n = 6 for the remaining groups). Results are presented as mean ± S.E.M. In b and e, *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001, one-way ANOVA plus Sidak’s multiple comparisons test. In d, g, i, j **p < 0.005, ****p < 0.0001, unpaired, two-tailed Kolmogorov–Smirnov test.