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Figure 4 | Molecular Medicine

Figure 4

From: RETRACTED ARTICLE: The α7 Nicotinic Acetylcholine Receptor Agonist GTS-21 Improves Bacterial Clearance in Mice by Restoring Hyperoxia-Compromised Macrophage Function

Figure 4

GTS-21 inhibits hyperacetylation of HMGB1 and cytoplasmic translocation of nuclear HMGB1 in macrophages. (A) RAW 264.7 cells were either exposed to 21% O2 or 95% O2 in the absence or presence of GTS-21 (50 µmol/L) for 24 h. HMGB1 localization was visualized by immunofluorescence microscopy with anti-HMGB1 antibody (red). Counterstaining with DAPI was used to visualize nuclei (blue). Bar = 10 µm. Multiple pictures were taken using an immunofluorescence microscope to visualize HMGB1. The immunofluorescence images shown are representative of three independent experiments. (B) Representative spectra of the liquid chromatography mass spectrometric (LC-MS) characterization of peptides produced from HMGB1 derived from macrophage cell lysates enzymatically cleaved with endopeptidase GluC. Macrophages were exposed to 95% O2 in the presence or absence of GTS-21. The presence of the peptides with molecular weights of 1,750 and 1,343 Da indicates the hyper-acetylation of lysine residues within NLS1 and NLS2, respectively. The presence of the peptides with molecular weights of 1,624 and 1,133 Da indicate the hypoacetylation of lysine residues within NLS1 and NLS2, respectively. (C) Representative spectra of the liquid chromatography tandem mass spectrometric (LC-MS/MS) characterization of a peptide (amino acids 180–188) covering the lysine (K) residues within NLS2 of HMGB1 to confirm the presence or absence of acetyl modifications on specific K residues. Acetyl modifications are represented as (ac) on specific lysine residues (K181, K182, K183 and K184) when required, and b and y ions are highlighted were appropriate.

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