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Proteomic analysis of FABP5 oxidative modifications in LPS-stimulated BMDMs under antioxidant treatment

ABSTRACT: This study aims to investigate the oxidative modifications of FABP5 in macrophages during inflammatory stimulation and how these changes are affected by antioxidant treatment. Bone marrow-derived macrophages (BMDMs) were pretreated with 5 mM N-acetylcysteine (NAC) or 250 μM Tempol for 2 hours, followed by stimulation with 500 ng/mL LPS for 30 minutes. Cytoplasmic proteins were extracted, FABP5 was immunoprecipitated, and the immunoprecipitated proteins were analyzed by LC-MS/MS to identify oxidative modifications of FABP5 and determine the regulatory effects of antioxidants under inflammatory conditions.

ORGANISM(S): Mus Musculus

SUBMITTER: Shenhai Gong

PROVIDER: PXD067487 | iProX | Tue Aug 19 00:00:00 BST 2025

REPOSITORIES: iProX

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Project description:Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of lipid-loaded macrophages in the arterial wall. Intimal macrophages internalize modified lipoproteins such as oxidized LDL (oxLDL) through scavenger receptors, leading to storage of excess cholesteryl esters in lipid bodies and a "foam cell" phenotype. In addition, stimulation of macrophage Toll-like receptors (TLRs) has been shown to promote lipid body proliferation. We investigated the possibility that there are transcriptional regulators that are common to both pathways for stimulating foam cell formation (modified lipoproteins and TLR stimulation), and identified the transcription factor ATF3 as a candidate regulator. In this specific microarray study, we re-analyzed a subset of the data from a 2006 microarray experiment (Gilchrist et al., Nature, 441:173-178, 2006) in which wild-type and Atf3-/- murine macrophages were stimulated with LPS. The goal of this analysis was to identify genes whose LPS responses are significantly affected by loss of ATF3 in macrophages, using up-to-date genome annotations. The raw microarray data files included in this submission were originally released to the public as a part of a larger microarray dataset that accompanied the Gilchrist et al. study (ArrayExpress accession # E-TABM-102). These data were re-analyzed using the CustomCDF v13 ENSG probesets as a part of a transcriptomic study of the response of macrophages to stimuli, including LPS, that induce foam cell formation (see GSE32358 and GSE32359). In the Gilchrist study, the experiment design was as follows: Female mice of the indicated strains were sacrificed at 8-12 weeks of age, and macrophages were derived from the femoral bone marrow using rhM-CSF. On day six, macrophages were plated and LPS introduced into the medium as indicated. After four hours, RNA was isolated using Trizol. Labeled cRNA derived from the RNA samples was hybridized to Affymetrix Mouse Genome 430 2.0 GeneChips. For the current study, probe-level instensity data were re-processed using gene-level probesets from the CustomCDF project (ENSG, v13), and are in log2 scale.

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Proteomic analysis of FABP5 oxidative modifications in LPS-stimulated BMDMs under antioxidant treatment

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