Western blot analysis of caspase-1 and IL-1β

Frozen clarified abscess homogenates were thawed rapidly at 37°C and protein concentration measured by A₂₈₀ (Nanodrop 1000 Spectrophotometer; ThermoFisher Scientific, Wilmington, DE). Equal amounts of total protein were separated by SDS-PAGE on a 4–12% Bis-Tris BOLT gel in MES-SDS running buffer (Life Technologies, Grand Island, NY), prior to transfer to nitrocellulose membranes. Membranes were blocked with 3% non-fat milk (NFM) in TBS (20 mM Tris, pH 7.5 and 150 mM NaCl) for 1.5 h at 22°C, then probed overnight at 4°C with rabbit anti-mouse anti-pro Caspase 1 + p10 + p12 antibody (Abcam, Cambridge, MA) or rabbit anti-mouse IL-1β (Abcam) in TBS plus 1% NFM. After washing with TBST (TBS with 0.1% Tween 20), membranes were developed using SuperSignal™ Maximum Sensitivity Substrate (ThermoFisher Scientific) following incubation with goat anti-rabbit IgG poly-horseradish peroxide–conjugated secondary antibody (ThermoFisher Scientific). Imaging was performed using a Protein Simple FluorChem R imaging system (Protein Simple, Santa Clara, CA) and band intensity determined using Image Studio Lite software ( LI-COR Biosciences, Lincoln, NE) relative to total protein loaded based on SYPRO® Ruby staining (Lonza, Allendale, NJ).