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. 2024 Jan 2;15:45. doi: 10.1038/s41467-023-44364-y

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© The Author(s) 2024

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. 1 — a FASN activity in patient-derived fibroblasts (index) and age-and sex matched controls (control) estimated by incorporation of ¹⁴C-acetate into lipid fraction of fibroblasts (data are presented as mean ± SEM, n = 6, unpaired two-tailed Student’s t test *P = 0.0022). b Western Blot against HA, FASN and LDHA of cycloheximide (CHX) incubated HEK293T-cells expressing HA-tagged wild type FASN (WT) or HA-tagged patient variant R2177C-FASN (R2177C) and (c) western blot quantification (data are presented as mean ± SEM, n = 3, unpaired two-tailed Student’s t test *P = 0.0094). d Western blot against HA, FASN and ubiquitin of HA-immunoprecipitated (HA I.P.) HA-tagged wild-type FASN (WT) or HA-tagged R2177C FASN (R2177C) from overexpressing HEK293T-cells treated with (+) or without (−) the proteasome inhibitor MG132. (e) Volcano plot showing significantly differentially abundant ubiquitinylated peptides between immunoprecipitated FASN from WT (WT) and R2177C-FASN (FASN^R2177) cells after incubation with MG132 (n = 4; two-way ANOVA highlighted are peptides with corresponding P-value < 0.05 and fold change difference > 1.5). f Western blot against HA, FASN, acetyl-lysine (Ac-Lys) and phospho-serine (P-Ser) of HA-tag immunoprecipitation (HA I.P.) in HEK293T cells overexpressing HA-tagged wild-type FASN (WT) or HA-tagged R2177C-FASN (R2117C) constructs. Western blots are representative picture of three independent experiments with the same result. g HA-tagged wild-type FASN (WT) or R2177C-FASN (R2177C) was overexpressed in HEK293T cells and after HA-immunoprecipitation (HA I.P.), WT and variant FASN was incubated with (+) or without (−) acetyl-CoA (Ac-CoA) in vitro for 6 h at 37 °C and western blot against HA, FASN, and acetyl-lysine (Ac-Lys) was performed. Western blots are representative picture of three independent experiments with the same result. h Western blots against HA, FASN and acetyl-lysine (Ac-Lys) of HA-tag immunoprecipitation (HA I.P.) in HEK293T cells expressing HA-tagged wild-type FASN (WT) or R2177C variant FASN (R2177) and HDAC3, a FASN deacetylase, co-treated with (+) or without (−) the proteasome inhibitor MG132. Western blots are representative picture of three independent experiments with the same result. i Ubiquitinylated FASN peptide abundance of immunoprecipitated R2177C-FASN overexpressed in HEK293T-cells in combination with a control (R2177C + empty vector) or HDAC3-vector (R2177C + HDAC3) (n = 4, data are shown as fold change vs. R2177C + empty vector). Source data are provided as a Source Data file.