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. 2023 Aug 17;14:4987. doi: 10.1038/s41467-023-40597-z

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© The Author(s) 2023, corrected publication 2023

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. 5 — a AML12 cells were transfected with the plasmids encoding PAK4 or PAK4^S474A, along with NCoR1, as indicated. Phosphorylation of NCoR1 was analyzed by immunoblotting of NCoR1 after immunoprecipitation with a p-Ser/Thr antibody. b Recombinant NCoR1 was incubated with active PAK4 and [³²P] ATP for 30 min at 31 °C. Proteins in the mixture were resolved by SDS-PAGE and visualized by autoradiography. Loading of proteins was confirmed by Coomassie blue staining. c In vitro peptide-competing kinase assay using synthetic oligopeptides comprising putative PAK4 target sites (P1: S837, P2: T1619, or P3: T2124) was performed. The diagram shows the sequence alignment of the putative phosphorylation sites of NCoR1 in various mammalian species. d PPRE-luciferase activities were measured in HEK293T cells co-transfected with PAK4, PPARα, and either wild-type (WT) or mutated NCoR1 (S837A, T1619A, T2124A, or T1619A/T2124A) (n = 6). e Co-IP and immunoblot analysis in HEK293T cells after co-transfection of NCoR1 (WT or T1619A/T2124A) and PAK4. Whole-cell lysates (WCL), nuclear extracts (NE), and cytosolic extracts (CE) were analyzed for NCoR1 protein levels and quantified (n = 3). f Ubiquitination of NCoR1 in HEK293T cells after co-transfection of HA-Ub and NCoR1 (WT or T1619A/T2124A). g Phosphorylation and ubiquitination of NCoR1 were analyzed in liver tissues from fed- or 24 h-fasted Pak4 LKO or WT mice. Protein levels of the indicated proteins were also analyzed in the WCL, NE, and CE. h–j Eight-week-old male C57BL/6 mice were injected with either adenoviruses expressing NCoR1 (AdNCoR1^WT or AdNCoR1^{T1619A/T2124A}), PAK4 (AdPAK4), or LacZ (AdLacZ), and were subsequently fasted for 24 h. Blood levels of βOHB (h, n = 4), gross liver morphology, and microscopic analysis (Oil Red O and H&E stain) of liver tissues (i, scale bars, 100 µm), as well as TG levels in the liver (j, n = 5), were compared. RLU relative luminescence unit, ns no significance. Data are presented as the mean ± SEM. Unpaired two-tailed t test was conducted for statistical analyses (d, e, h, j). ^*P < 0.05 and ^**P < 0.01. Source data are provided as a Source Data file.