Macrophage Protein Expression Analysis

Macrophages were harvested and resuspended in RIPA lysis buffer (GenDEPOT, Baker, TX, USA). Concentrations of proteins were determined using a BCA protein assay (ThermoScientific, Rockford, IL, USA). Proteins (30 μg) were resolved by 10% SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose. Membranes were blocked in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) and 5% skim milk, incubated with specific primary antibodies recognizing β-actin, COX-2, iNOS, arginase-1, caspase-1, and caspase-11, and then incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA, USA). Signals were developed using an enhanced chemiluminescence system (Pierce Biotechnology Inc., Rockford, IL, USA) [14 (link)].