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. 2022 Jul 8;13:3972. doi: 10.1038/s41467-022-31735-0

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© The Author(s) 2022

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. 3 — A PAS-reactive phosphorylation of a C-terminal fragment TRIM24^Cter. GFP-TRIM24^Cter (spanning from G601-end of TRIM24) was expressed in HEK293 cells stimulated with or without insulin after pre-treatment with PI-103. PAS-reactive phosphorylation was detected on immunoprecipitated GFP-TRIM24^Cter using the PAS antibody. Total and phosphorylated PKB were determined in cell lysates. B Diagrammatic illustration of the Ser¹⁰⁴³ site and two NLSs on TRIM24. Ser¹⁰⁴³ is highlighted in red. C Effects of Ser¹⁰⁴³ mutation on insulin-induced PAS-reactive phosphorylation of TRIM24. WT GFP-TRIM24 and mutant GFP-TRIM24^S1043A were expressed in HEK293 cells that were stimulated with or without insulin. After immunoprecipitated from cell lysates, phosphorylation of TRIM24 was determined using the PAS antibody. D In vitro phosphorylation of WT GST-TRIM24 and mutant GST-TRIM24^S1043A by a recombinant PKBα. Phosphorylated GST-TRIM24 was detected using the PAS antibody. E Ser¹⁰⁴³ phosphorylation on GFP-TRIM24 in response to insulin. WT GFP-TRIM24 and mutant GFP-TRIM24^S1043A were expressed in HEK293 cells that were stimulated with or without insulin. Ser¹⁰⁴³ phosphorylation on GFP-TRIM24 was determined using the site-specific phospho-antibody. F Ser¹⁰⁴³ phosphorylation on endogenous TRIM24 in response to insulin. Mouse primary hepatocytes were stimulated with or without insulin. Endogenous TRIM24 was immunoprecipitated from cell lysates and Ser¹⁰⁴³ phosphorylation on it was determined using the site-specific phospho-antibody. Quantitation results were shown in Supplementary Fig. 1E. G Subcellular distribution of GFP-TRIM24 WT, S1043A and S1043D mutants. GFP-TRIM24 proteins were expressed HEK293 cells, and their subcellular distribution was determined in the nuclear and cytosolic fractions via immunoblotting. GAPDH was used as a cytosolic marker while Lamin A/C was detected as a nuclear marker. H Subcellular localisation of GFP-TRIM24 WT and mutant proteins in response to insulin. GFP-TRIM24 WT, S1043A and S1043D mutants were expressed mouse primary hepatocytes that were stimulated with or without insulin. After fixation, cells were stained with DAPI and photographed using a confocal microscope. Scale bars indicate 10 μm in length. Source data are provided as a Source Data file.