Journal: Cellular and Molecular Life Sciences: CMLS
Article Title: Enhancement of mycobacterial pathogenesis by host interferon-γ
doi: 10.1007/s00018-024-05425-7
Figure Lengend Snippet: Direct interaction of M. bovis BCG and M. tuberculosis with interferon-γ. ( A ) Specific binding of IFNγ to M. bovis BCG in a dose-dependent manner. Mycobacteria were fixed, incubated with different concentrations of IFNγ, heat inactivated IFNγ (HI-IFNγ), BSA, IL12, IL1β, and IL18, and analyzed by ELISA. ( B ) Immunofluorescence demonstrated IFNγ binding to M. bovis BCG. Mycobacteria were fixed, blocked, incubated with 10 µg/ml BSA or cytokines, immunostained, and analyzed by fluorescence microscopy. Scale bar, 1 μm. Arrow indicates protein colocalization. ( C ) Immunoblotting analysis demonstrated IFNγ binding to M . bovis BCG and M. tuberculosis . Bacteria were fixed, blocked, incubated with 10 µg/ml BSA or IFNγ, washed and analyzed by immunoblotting with antibodies against IFNγ and mycobacterial RpsO. Recombinant mouse IFNγ (10 ng) was used as a positive control. Data information: Statistical analysis in Fig. 1A was performed with two-way ANOVA followed by multiple comparisons among groups. The results are the mean values ± standard deviations of three biological replicates each with three technical replicates. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns , not significant
Article Snippet: Ten days after the final injection, serum samples were obtained, and ELISA was performed to determine the antibody titer; recombinant mouse IFNγ protein (#ab259378), recombinant mouse IL12 protein (#ab259419), recombinant mouse IL1β protein (#ab259421), recombinant mouse TNFα protein (#ab259411), and TMB ELISA substrate (#ab171523) from Abcam; mouse monoclonal anti-β-actin antibody (#3700S) from Cell Signaling; recombinant mouse IFNγ protein (#IF005) and glass bead (#G8772) from Sigma; recombinant mouse IL1β protein (#BMS332), recombinant mouse IL18 protein (#PMC0184), IFNγ rabbit monoclonal antibody (#701121), IFNγ monoclonal antibody (clone XMG1.2), eBioscience (#14-7311-81), rat anti-mouse IL12 p70 (clone 9A5) (#ENMM120), rabbit anti-mouse IL1β (#500-P51), rabbit anti-mouse IL18 (#210-401-323 S), rat IgG1 kappa isotype control (eBRG1), eBioscience (#14-4301-82), goat anti-rabbit IgG-HRP (#31460), goat anti-rat IgG-HRP (#31470), geneticin selective antibiotic (G418 Sulfate) (#10131035), and fluoromount-G mounting medium (#00-4958-02) from Thermo Fisher Scientific; FITC anti-mouse IFNγ antibody (#505806), FITC goat anti-rat IgG antibody (#405404), and Alexa fluor 647 anti-mouse IgG1 antibody (#406618) from BioLegend; rat anti-mouse IFNγ receptor 1 (CD119) (clone GR-20) and rat IgG2a isotype control (clone 2A3) from BioXCell; and heat shock protein 65 (mycobacterial) monoclonal antibody (clone 4H11) (#ADI-SPA-882-E) from Enzo Life Science.
Techniques: Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Fluorescence, Microscopy, Western Blot, Bacteria, Recombinant, Positive Control