Journal: The Journal of Biological Chemistry

Article Title: Involvement of langerin in the protective function of a keratan sulfate-based disaccharide in an emphysema mouse model

doi: 10.1016/j.jbc.2023.105052

Figure Lengend Snippet: CapG was identified as a langerin-binding molecule and localized in the nucleus. A , silver staining of the SDS-PAGE gel. The immunoprecipitates prepared from BMDCs with an anti-langerin antibody and a non-relevant IgG separated in SDS-PAGE were then subjected to silver staining. The dashed square area was cut out and the contents analyzed by mass spectrometry. B , Western blotting of the immunoprecipitates prepared from BMDCs with an anti-langerin antibody and a non-relevant IgG. The membrane was blotted with an anti-CapG antibody. Arrow indicates CapG. C , subcellular localization of CapG in BMDCs examined by western blotting. Nuclear-rich fraction and cytoplasm-rich fraction were prepared and analyzed. Lamin B1 was used as a maker for nuclear fraction and GAPDH was used as a marker for cytoplasmic fraction. D , subcellular localization of CapG in HL-60, THP-1, and U937 cells examined by western blotting. E , immunocytochemical staining of THP-1 and U937 cells with an anti-CapG antibody and DAPI. Red indicates CapG and blue indicates nuclear. Bars indicate 10 μm. Images were pictured under confocal microscopy. F , subcellular localization of CapG in BMDCs after incubation with L4 (100 μg/ml) for 2 days examined by western blotting. GAPDH was used as a marker for the cytoplasmic fraction. Independent experiments were performed and the normalized nuclear localization of CapG after incubation with or without L4 is shown in the right panel. ∗ p < 0.05.

Article Snippet: The protein concentration was measured by using BCA protein assay kit (BioRad Laboratories) and a part of lysate including 2 mg of total protein was incubated with 4 μg of anti-CapG antibody (Santa Cruz Biotechnology, Inc, catalog number sc-166428) or non-relevant isotype IgG (Santa Cruz Biotechnology, Inc) overnight at 4 °C under rotation.

Techniques: Binding Assay, Silver Staining, SDS Page, Mass Spectrometry, Western Blot, Membrane, Marker, Staining, Confocal Microscopy, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Involvement of langerin in the protective function of a keratan sulfate-based disaccharide in an emphysema mouse model

doi: 10.1016/j.jbc.2023.105052

Figure Lengend Snippet: CapG functioned as an inhibitory transcription factor to suppress TNFa gene expression . A , ChIP-qPCR analysis targeting the promoter or enhancer region for the IL-6 , TNFα , and Mcp-1 genes. Genomic DNA of BMDCs was fragmentated and immunoprecipitated with an anti-CapG antibody or a non-relevant IgG, then, immunoprecipitated DNAs were purified and analyzed by qPCR analysis. Relative DNA content relative to input samples were indicated as the mean±SD. N.D.: not detected. ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, ns: not significant. TF: transcription factor binding site. B , Western blot analysis of CapG in clonal cell lines derived from THP-1 cells. After transfection of a vector to knockout CAPG gene by genome editing in THP-1 cells, four clonal cell lines were established and analyzed. GAPDH was used as a loading control. C , the gene expression level of TNFα , IL-1β , and MCP-1 in CAPG knockout cells ( CAPG -KO) and control cells were examined by qRT-PCR analysis. Before the analysis, the cells were differentiated by incubation with PMA (phorbol 12-myristate 13-acetate) (20 nM) for 2 days. The expression levels were normalized to GAPDH genes. ∗∗ p < 0.01, ns: not significant.

Article Snippet: The protein concentration was measured by using BCA protein assay kit (BioRad Laboratories) and a part of lysate including 2 mg of total protein was incubated with 4 μg of anti-CapG antibody (Santa Cruz Biotechnology, Inc, catalog number sc-166428) or non-relevant isotype IgG (Santa Cruz Biotechnology, Inc) overnight at 4 °C under rotation.

Techniques: Gene Expression, ChIP-qPCR, Immunoprecipitation, Purification, Binding Assay, Western Blot, Derivative Assay, Transfection, Plasmid Preparation, Knock-Out, Control, Quantitative RT-PCR, Incubation, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Involvement of langerin in the protective function of a keratan sulfate-based disaccharide in an emphysema mouse model

doi: 10.1016/j.jbc.2023.105052

Figure Lengend Snippet: L4 mitigates the inflamed state through the langerin-CapG axis in the lung of the emphysema mouse model. A , representative images of the staining for CD80 in the lung sections of wild type and langerin -KO mouse after administration of L4 and elastase. Arrowheads indicate CD80-positive cells. The number of CD80-positive cells were counted in random 20 fields in each mouse. ∗∗∗∗ p < 0.0001. Bars indicate 50 μm. B , representative images of the staining for TNFα in the lung sections of wild type and langerin -KO mouse after administration of L4 and elastase. The number of cells expressing TNFα in cytoplasmic region were counted in 20 randomly selected fields in each mouse and the ratio against the total cell number was shown. ∗∗∗∗ p < 0.0001. Bars indicate 20 μm. C , representative images of the staining for CapG in the lung sections of wild type and langerin -KO mouse after administration of L4 and elastase. The number of cells expressing CapG in nuclear were counted in 20 randomly selected fields in each mouse and the ratio against total CapG positive cells was shown. ∗∗∗∗ p < 0.0001. Bars indicate 20 μm.

Article Snippet: The resulting sections were incubated with an anti-CD80 antibody (Santa Cruz Biotechnology, Inc, catalog number sc-376012), an anti-TNFα antibody (Novus Biologicals, Littleton, CO, catalog number NBP1-19532SS), or an anti-CapG antibody (Santa Cruz Biotechnology, Inc, catalog number sc-166428).

Techniques: Staining, Expressing