Goat anti mouse immunoglobulin

Tissues were lysed using radioimmunoprecipitation assay (RIPA) lysis buffer containing a mixture of protease inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Adenoid protein samples (30 μg) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was first incubated with primary antibodies against IL-17C and GAPDH (Abcam, Cambridge, UK), washed, and then incubated with secondary antibodies, namely horseradish peroxidase-labeled goat anti-rabbit or goat anti-mouse immunoglobulin (Jackson Labs, Bar Harbor, ME, USA). Signals were detected using the Enhanced Chemiluminescence detection reagent (Amersham, Buckinghamshire, UK) and relative band intensities were calculated using the Image J software (National Institutes of Health, Bethesda, MD, USA). The total number of adenoid tissues analyzed in each western blot assay is indicated in the corresponding figure legend.

Serum levels of polyclonal IgM and IgG and anti-chromatin IgG subclasses were determined in individual mice by triplicate ELISA analysis using 96-well plates coated with goat anti-mouse immunoglobulin (Jackson ImmunoResearch Laboratories) or chromatin, respectively. Bound antibodies were detected using alkaline phosphatase-conjugated goat antibodies to mouse IgM, IgG or IgG subclasses (Southern Biotech), and standard curves were generated using calibrated mouse serum (Nordic Immunology). Commercial ELISA kits were used to examine IFN-α (PBL InterferonSource), TNF-α or IL-6 (Biolegend) levels in mouse sera and supernatants of B cell and DC cultures.