Journal: Nature Communications

Article Title: NMI and IFP35 serve as proinflammatory DAMPs during cellular infection and injury

doi: 10.1038/s41467-017-00930-9

Figure Lengend Snippet: NMI stimulates macrophages through the TLR4 pathway. a Western blot analysis of mNMI in the BMDM cell lysate after 1 h incubation with recombinant mNMI. The BMDM cells were isolated from Nmi −/− mice and pretreated with macrophage colony-stimulating factor (MCSF). b , c TNF and IL-6 released by BMDMs from WT (C57BL/6) mice, pretreated with bafilomycin A1 (10 nM), TAK-242 (100 nM) or dimethyl sulphoxide (DMSO) for 2 h and stimulated with mNMI (5 μg ml −1 ) or LPS (100 ng ml −1 ) for 8 h. d – g TNF and IL-6 levels in the supernatants of BMDMs from WT, Tlr4 −/− and Tlr2 −/− mice were analyzed using ELISA 4 h post activation by different stimulus. h After incubation with mNMI-GFP for 1 h, the percentage of GFP labeled CD11b + F4/80 + cells was determined by Flow cytometric analysis. The cells were isolated from spleen in WT or Tlr4 −/− mice. i NMI in the human THP1 cell (ATCC TIB-202™) lysates interacts with hTLR4. Ni-NTA beads coupled with 2 μg His-hTLR4 and/or His-hMD2 fusion proteins were used as bait. j The luciferase activity of HEK293T cells (ATCC CRL-11268™) are shown after stimulated with 5 μg ml −1 mNMI for 4 h (in the presence or absence of 25 μg ml −1 polymyxin B (PMB)). The cells were pre-transfected with mTLR4-MD2-CD14 and NF-κB promoter with luciferase activity. 100 ng ml −1 LPS was administrated as positive control. In b – e and g , error bars indicate ± s.e.m. from 3 biological replicates. Significance was tested by one-way ANOVA followed by Student–Newman–Keuls test. ** P < 0.01

Article Snippet: The total RNA was extracted from HEK293T (ATCC CRL -11268TM) and BMDM cells using Trizol reagent (InvivoGen, 15596018) according to the manufacturer’s instructions.

Techniques: Western Blot, Incubation, Recombinant, Isolation, Enzyme-linked Immunosorbent Assay, Activation Assay, Labeling, Luciferase, Activity Assay, Transfection, Positive Control

Journal: Nature Communications

Article Title: NMI and IFP35 serve as proinflammatory DAMPs during cellular infection and injury

doi: 10.1038/s41467-017-00930-9

Figure Lengend Snippet: IFP35 serves as a DAMP and stimulates the inflammatory responses. a , b Immunoblotting a and ELISA analysis b of IFP35 in the supernatant and cytoplasmic fraction of mouse RAW264.7 cells (ATCC TIB-71™), treated with LPS (1 μg ml −1 ). c , d Mice ( n = 5 for each group) were intraperitoneally injected with LPS from E. coli (100 ng ml −1 , c ) or live S. typhimurium (1 × 10 4 CFU per mouse, d ). The serum concentrations of IFP35 in mice were determined by ELISA. Data are presented as the mean ± s.e.m. Significance was tested by unpaired Student’s t -test (** P < 0.01). e TNF level in the supernatants of human THP1 cells (ATCC TIB-202™) was analyzed by ELISA 8 h post incubation with LPS (100 ng ml −1 ) or different purified proteins (5 μg ml −1 respectively). Data are presented as the mean ± s.e.m of three independent experiments. ** P < 0.01. f The luciferase activity of HEK293T cells (ATCC CRL-11268™), transiently transfected with the genes of hTLR4–MD2–CD14 and NF-κB promoter with luciferase, and stimulated with 5 μg ml −1 hIFP35ΔN for 4 h (in the presence or absence of 25 μg ml −1 polymyxin B (PMB)). 100 ng ml −1 LPS was administrated as positive control. Data are presented as the mean ± s.e.m. of three independent transfections and are given as the ratio of luciferase activity relative to unstimulated cells. Significance in d and e was tested by one-way ANOVA followed by Student–Newman–Keuls test. ** P < 0.01. g The survival rate of wild type ( n = 14) and Ifp35 −/− mice ( n = 14) intraperitoneally injected with LPS (50 μg kg −1 ) and d -gal (1 g kg −1 ). Significance was tested by log-rank test.* P < 0.05

Article Snippet: The total RNA was extracted from HEK293T (ATCC CRL -11268TM) and BMDM cells using Trizol reagent (InvivoGen, 15596018) according to the manufacturer’s instructions.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Injection, Incubation, Purification, Luciferase, Activity Assay, Transfection, Positive Control

Journal: bioRxiv

Article Title: Integrative Structural Modeling of Intrinsically Disordered Regions in a Human HDAC2 Chromatin Remodeling Complex

doi: 10.1101/2025.08.08.669391

Figure Lengend Snippet: A . Nuclear Localization of HALO-C16orf87 in HEK293T. Halo-tagged proteins are labeled with HaloTag® TMR ligand (red). Nuclei are stained with Hoechst dye (blue). B . Heatmap plotting relative protein abundance expressed as log2FC with the hierarchical bi-clustering of the 8 Halo-tagged baits: C16orf87, MIER1b, MIER1-2, HDAC1, HDAC2, SIN3A, SUDS3, SAP30, with the brightest yellow indicating high log2FC (Suppl. Table S1A). C . A network analysis was performed using Cytoscape on members of HDAC1/2 associated complexes significantly enriched with at least one of the bait proteins compared to controls (log2FC > 3, FDRup< 0.05; Suppl. Table S1A). The Halo-tagged bait proteins (C16orf87, MIER1b, MIER1-2, HDAC1, HDAC2, SIN3A, SUDS3, and SAP30) are source nodes in gray, with SIN3A associated proteins in pink, NuRD associated proteins in light blue, CoREST associated proteins in green, MIER associated proteins in purple, HDAC1/2 (in light yellow/orange, and C16orf87 in red. D . Representative images of acceptor and donor fluorescence for each sample in the AP-FRET experiments. SNAP-F-C16orf87 is labeled with the SNAP-Cell® 505-Star ligand, while HDAC1/2-Halo are labeled with the HaloTag® MR ligand (see data in Suppl. Table S2A).

Article Snippet: HEK293T cells (ATCC1CRL-11268TM) were purchased from American Type Culture Collection (ATCC).

Techniques: Labeling, Staining, Quantitative Proteomics, Fluorescence

Journal: bioRxiv

Article Title: Integrative Structural Modeling of Intrinsically Disordered Regions in a Human HDAC2 Chromatin Remodeling Complex

doi: 10.1101/2025.08.08.669391

Figure Lengend Snippet: A. Nuclear localization of SNAP-Flag-C16orf87 in Flp-In TM -293 cells stably expressing HDAC1-Halo or HDAC2-Halo. Halo-tagged proteins are labeled with HaloTag® TMR ligand (red); SNAP-tagged proteins are labeled with SNAP-Cell ® 505-Star ligand (green); nuclei are stained with Hoechst dye (blue). Nuclear localization was also observed with a Halo-tagged C16orf87 construct . B. Bait-specific hierarchical clustering on Topological scores (TopS) including the 8 Halo-tagged baits: C16orf87, MIER1b, MIER1-2, HDAC1, HDAC2, SIN3A, SUDS3, and SAP30. Red corresponds to high TopS values and blue corresponds to negative TopS values (Suppl. Table S1B). C. AP-FRET analysis between SNAP-F-C16orf87 (labeled with SNAP-Cell® 505-Star ligand) and HDAC1/2-Halo (labeled with HaloTag® MR ligand). The positive control was a Halo-NLS-SNAP construct to measure the FRET efficiency of Halo and SNAP when close together; the negative control confirmed an absence of FRET in cells expressing NLS-Halo and NLS-SNAP on their own. Unpaired t-tests were used for statistical analyses where ***: p≤ 0.0001. Representative images of acceptor and donor fluorescence are provided in , while all measurements and Image J processing are reported in Suppl. Table S2A. D. Deacetylase activity assays were performed using equal volumes of eluates from the Halo-only, HDAC2-Halo, and Halo-C16orf87 affinity purifications. Deacetylase activity was measured by a colorimetric reaction, and relative fluorescence unit (RFU) values were normalized to total protein content in each sample as measured by BCA protein assay. Assays were performed in triplicate with error bars representing SEM. Experimental workflow, acquisition setting, raw measurements, and calculations are reported in Supp. Table S2B. E. HEK293T cell lysates expressing tagged C16orf87 with or without 7 Halo-tagged baits (MIER1b, MIER1-2, HDAC1, HDAC2, SIN3A, SUDS3, and SAP30) were used for Halo pulldown analysis. Equal volumes of eluate were analyzed by SDS PAGE and Western blotting. SNAP-FLAG-C16orf87 was detected using anti-FLAG mouse monoclonal primary antibody and IRD 680 LT labeled goat anti-Mouse secondary antibody. Halo-tagged baits were detected using anti-Halo rabbit polyclonal primary antibody and IRD 800CW labeled goat anti-Rabbit secondary antibody. F. Quantification of relative C16orf87 enrichment in HDAC1/2 and MIER protein pulldowns. Normalized values account for differences in C16orf87 concentration between Halo-tagged samples determined by quantitative Western blotting (Suppl. Table S2C). Band intensities were quantitated using Image Studio v5.2 (Li-Cor). Unpaired t-tests were performed for statistical analyses where *** = p≤0.0001.

Article Snippet: HEK293T cells (ATCC1CRL-11268TM) were purchased from American Type Culture Collection (ATCC).

Techniques: Stable Transfection, Expressing, Labeling, Staining, Construct, Positive Control, Negative Control, Fluorescence, Histone Deacetylase Assay, Activity Assay, Bicinchoninic Acid Protein Assay, SDS Page, Western Blot, Concentration Assay