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. 2020 Apr 27;11:2039. doi: 10.1038/s41467-020-15995-2

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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. 4 — a A Proteome Profiler assay (R&D Systems) was performed with GapmeR-transfected human cardiomyocytes to assess levels and phosphorylation status of apoptosis-related proteins upon SARRAH knockdown. Black bars depict downregulated NRF2 target genes (n = 3; SEM; *t-test p = 0.001 for Catalase, p = 0.0046 for PON2, p = 0.016 for Bad, p = 0.017 for Bcl-x, p = 0.024 for Clusterin, p = 0.026 for cleaved caspase-3). b NRF2 mRNA levels were measured by qRT-PCR in human cardiomyocytes after transfection with GapmeRs to silence SARRAH or control GapmeRs (top panel; n = 4; SEM; *t-test p = 0.014). NRF2 protein levels were measured by Western blotting. GAPDH served as loading control (n = 3). c Reactive oxygen species were measured using a CM-H2DCFDA probe in human cardiomyocytes after transfection with GapmeRs to silence SARRAH or control GapmeRs (n = 4; SEM; *t-test p = 0.021). d Caspase-3/7 activity was measured in human cardiomyocytes that lentivirally overexpress NRF2 or mock-transduced cells that were transfected with either control GapmeRs or GapmeRs targeting SARRAH (n = 3; SEM; *two-way ANOVA F = 6.8, p = 0.0266). e Scheme depicting the RNA pulldown approach used to identify proteins interacting with endogenous Sarrah. 200 pmol of biotinylated scrambled oligo or two biotinylated Sarrah antisense oligos were added to HL-1 cell lysate, coupled to streptavidin beads and eluted. f Sarrah pulldown efficiency was determined by qRT-PCR of eluted samples (displayed as % input; n = 5; SEM; *t-test p = 0.0317). g Volcano plot showing all proteins identified by mass spectometry analysis that are enriched in Sarrah pulldown as compared to pulldown with a scrambled oligo. CRIP2, the hit with the highest enrichment, is highlighted. h RNA-immunoprecipitations with antibodies against CRIP2 (rabbit antibody), p300 (mouse antibody) and histone acetylation H3K27ac (rabbit antibody) were performed in primary human cardiomyocytes. SARRAH levels were measured by qRT-PCR (n = 4, 5 and 8; SEM; *t-test p = 0.029 for p300; one-way ANOVA for CRIP2 p = 0.024; IgG immunoglobulin G).