Journal: Nature Communications

Article Title: Chromatin organization drives the search mechanism of nuclear factors

doi: 10.1038/s41467-023-42133-5

Figure Lengend Snippet: a paSMT is carried out using highly inclined laminated optical sheet (HILO) microscopy (top left) by labelling the endogenous HaloTag-p53 with the photoactivatable dye PA-JF 549 (bottom left). Movies, acquired at a framerate of 100 fps highlight quasi-immobile chromatin-bound molecules (cyan arrowhead) and diffusing (purple arrowhead) ones (max proj = maximal projection over the entire movie; cyan dotted line indicates the cell nucleus, scale bar: 5 µm). b We use vbSPT to classify track segments into bound and diffusing components, and then focus on diffusing molecules, by computing diffusional anisotropy, by calculating the distributions of angles \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\theta$$\end{document} θ between consecutive jumps, and the fold-anisotropy metric, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f}_{180/0}$$\end{document} f 180 / 0 , calculated as the probability of observing a backward displacement \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$p(150^\circ \le \theta \le 210^\circ )$$\end{document} p ( 15 0 ∘ ≤ θ ≤ 21 0 ∘ ) over the probability of observing a forward displacement \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$p(-30^\circ \le \theta \le 30^\circ )$$\end{document} p ( − 3 0 ∘ ≤ θ ≤ 3 0 ∘ ) . c Different NFs display different diffusional anisotropy, with factors poorly localized in DNA-dense regions displaying lower anisotropy than factors enriched in DNA-dense regions. d Fold-anisotropy metric \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f}_{180/0}$$\end{document} f 180 / 0 as function of the distance run by the molecules. p53, CTCF, and H2B display high diffusional anisotropy at a spatial scale of ~100–150 nm, a signature of transient trapping of these molecules in traps of similar size ( n cells = 30, 30, 29, 14, 31, n angles = 59470, 62813, 180414, 26052, 26566 for HaloTag, p565, p53, CTCF, and Histone H2B respectively, error bars: s.e.m. estimated through boot-strapping). e Analysis of diffusional anisotropy in our SMT/mSIM data allows us to identify that the highest diffusional anisotropy occurs for molecules with slow instantaneous diffusion coefficients in regions at high chromatin density (same data as in Fig. ). Source data are provided as a Source Data file.

Article Snippet: The antibodies employed in this study were: mouse monoclonal anti-p53 DO-1 (Santa Cruz Biotechnology, cat. sc-126; 1:3000 dilution, incubated 1 h at RT), rabbit monoclonal anti-p21 (Abcam, cat. ab109520; 1:1000 dilution, incubated overnight at 4 °C), rabbit monoclonal anti-GAPDH (Abcam, cat. ab128915; 1:50,000 dilution, incubated 1 h at RT), rabbit monoclonal anti-NF-κB p65 (Cell Signaling cat. D14E12 XP®; dilution 1:1000), rabbit polyclonal anti-Histone H2B (Abcam cat. ab1790, dilution 1:5000), mouse monoclonal anti-HaloTag (Promega G921A, dilution 1:1000), mouse monoclonal anti-vinculin (Thermo-Fisher, cat. MA5-11690; 1:4000 dilution, incubated 1 h at RT).

Techniques: Microscopy, Diffusion-based Assay

Journal: Nature Communications

Article Title: A chemical catalyst enabling histone acylation with endogenous acyl-CoA

doi: 10.1038/s41467-023-41426-z

Figure Lengend Snippet: a Acetylation of histone H2BK120 in living cells. LieD-transfected HEK293T cells were treated with the indicated catalysts (100 μM) for 10 h. Histone proteins were acid-extracted, and H2BK120 acetylation and total H2B subunit were detected with anti-H2BK120ac and anti-H2B antibodies, respectively, by western blot analysis. Dividing lines have been used to indicate where noncontiguous sections of a gel have been aligned for ease of comparison. Representative data from two independent experiments are shown. The acetylation yields at H2BK120 were quantified by LC–MS/MS analysis. The mean chemical yields of two independent experiments are shown. Source data are provided as a Source Data file. b Schematic illustration of metabolic isotopic labeling of Ac-CoA using [U- 13 C]-glucose. The U refers to “universally labeled” (i.e., all carbons are 13 C). An asterisk denotes the position of 13 C-incorporation. c Detection of isotopic labeling at acetylated H2BK120. LieD-transfected HEK293T cells were incubated in a medium containing non-labeled glucose (upper) or [U- 13 C]-glucose (lower), and were treated with m BnA-TMP(gly 1 ) 4 . Acid-extracted histones were treated with propionic anhydride to propionylate non-acetylated lysine residues, digested by trypsin and Glu-C peptidases, and then analyzed by LC–MS/MS. The [M + 2H] 2+ parent peaks were analyzed. Isotopic distribution of the H2B G114-K125 peptide, whose lysine residues were all propionylated (i.e., non-acetylated, All Pr) are shown on the left, while that of the mono-acetylated peptide (K120ac) is shown on the right. As we observed [M + 2H] 2+ peaks, +2 Da shift was detected as +1 m / z shift, as shown in red (720.90 and 713.89). Representative data from two independent experiments are shown. Source data are provided as a Source Data file. d LC–MS/MS trace of the H2B G114-K125 K120ac peptide from LieD-transfected HEK293T cells cultured in [U- 13 C]-glucose and treated with m BnA-TMP(gly 1 ) 4 . The sequence and the calculated m / z values of the fragment ions are shown. Peaks shown in red indicated incorporation of +2 Da shift at H2BK120. Representative data from two independent experiments are shown. Source data are provided as a Source Data file.

Article Snippet: Rabbit polyclonal antibodies against H2B (abcam, ab1790, 1:2000 dilution for WB), H3K9ac (Merck, 07-352, 1:1000 dilution for WB), H3K18ac (abcam, ab1181, 1:1000 dilution for WB), and pan-K ma (PTM-Biolabs, PTM-901, 1:1000 dilution for WB), a rabbit monoclonal antibody against p300 (Cell Signaling Technology, 54062, 1:1000 dilution for WB), and mouse monoclonal antibodies against H2BK120ac (1 μg/mL for WB) and H3K27ac (Merck, 05-1334, 1:1000 dilution for WB) were used for western blot and ChIP assay.

Techniques: Transfection, Western Blot, Comparison, Liquid Chromatography with Mass Spectroscopy, Isotopic Labeling, Labeling, Incubation, Cell Culture, Sequencing

Journal: iScience

Article Title: Selective protein aggregation confines and inhibits endotoxins in wounds: Linking host defense to amyloid formation

doi: 10.1016/j.isci.2023.107951

Figure Lengend Snippet:

Article Snippet: Polyclonal rabbit antibodies against human histone 2b , Abcam , ab1790.

Techniques: Virus, Sterility, Recombinant, Mass Spectrometry, Activation Assay, Software

Journal: PLoS ONE

Article Title: Loss of the Histone Pre-mRNA Processing Factor Stem-Loop Binding Protein in Drosophila Causes Genomic Instability and Impaired Cellular Proliferation

doi: 10.1371/journal.pone.0008168

Figure Lengend Snippet: Protein lysates from wt, Slbp 15 , Slbp 10 , and H2aV 810 mutants were obtained from whole 3 rd instar larvae and probed with antibodies to A) H2b and H3, and B) H3K4-me2 and H3K9-me2. β-tubulin was used as a loading control for both panels.

Article Snippet: Polyclonal rabbit C-terminal H3 (1∶3000) (Abcam #1791), polyclonal rabbit H2b (Abcam #1790) (1∶3000), monoclonal mouse H3K9-me2 (Abcam #1220) (1∶750), and polyclonal rabbit H3K4-me2 (Abcam #7766) (1∶3000), and monoclonal mouse Β-tubulin (1∶1000) antibodies were obtained commercially.

Techniques:

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Combination of Coenzyme Q 10 Intake and Moderate Physical Activity Counteracts Mitochondrial Dysfunctions in a SAMP8 Mouse Model

doi: 10.1155/2018/8936251

Figure Lengend Snippet: Summary of used antibodies.

Article Snippet: H2B , Rabbit polyclonal anti-H2B , 1 : 1000 , Abcam.

Techniques:

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Combination of Coenzyme Q 10 Intake and Moderate Physical Activity Counteracts Mitochondrial Dysfunctions in a SAMP8 Mouse Model

doi: 10.1155/2018/8936251

Figure Lengend Snippet: Western blot analysis (a) and relative protein quantification (b) of PGC-1 α , TFAM, and SIRT5 expressed in tibialis anterior (TA) muscle, in sedentary (SED), physical exercise (PHY), ubiquinol (QH 2 ), and ubiquinol associated with physical exercise (QH 2 + PHY) mouse groups ( n = 5). PGC-1 α protein levels were normalized to H2B levels. TFAM and SIRT5 were normalized to VDAC1 levels. AU: arbitrary units. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗∗ p < 0.0001; (a) = vs. SED, (b) = vs. PHY, and (c) = vs. QH 2 .

Article Snippet: H2B , Rabbit polyclonal anti-H2B , 1 : 1000 , Abcam.

Techniques: Western Blot