Non immune igg control

Cell lysates were prepared using the RIPA Lysis Buffer System (Santa Cruz Biotechnology) and protein content concentrations were determined by Nano Drop 2000C spectrophotomer (Thermo Scientific). Immunoprecipitation was carried out using goat polyclonal anti-MFG-E8 antibody or non-immune IgG control (R&D Systems) and protein G-coupled magnetic beads according to the manufacture’s protocol (Life Technologies). Proteins were separated by standard SDS-PAGE on 10% acrylamide gels (Life Technologies) and transferred to polyvinylidene difluoride membrane (Bio-Rad) by electroblotting. The membranes were incubated in blocking buffer (5% nonfat dried milk, 10 mM Tris [pH 7.5], 100 mM NaCl, and 0.05% Tween 20) followed by probing with goat polyclonal anti-MFG-E8 antibody (R&D Systems) or anti-MFG-E8 mAb (18A2-G10; MBL) and visualization with horseradish peroxidase-conjugated secondary antibody and chemiluminescence using the Amersham Biosciences ECL system. Images were captured using a FluorChem M imaging system (ProteinSimple).

A neutralizing mAb to IL-17A (rat IgG2a; M210) was generously provided by Amgen, and azide-free rat IgG2a (RTK2758; Biolegend) served as isotype control. Purified polyclonal goat IgG antibody to IL-23p19 subunit and non-immune IgG control were purchased from R&D Systems. The antibodies or their controls were microinjected locally into the murine palatal gingiva (5 μg per site), three times weekly, using a 28.5-gauge MicroFine needle (BD Biosciences). Microinjections were performed on the mesial of the first molar and in the papillae between first and second and third molars on both sides of the maxilla (19 (link)).