Macrophages play a critical role in innate immunity through Toll-like receptor (TLR) signaling. Lipopolysaccharides (LPS) are a ligand of microbial origin that can trigger cell signaling in macrophages through TLRs and production of pro-inflammatory cytokines. Statin, a hypercholesterolemia drug, on the contrary, can reduce inflammatory cytokine production, and inflammation at large. Discovery-based quantitative proteomics is a useful method for unraveling complex protein networks and inter-protein interactions. Here, we describe protocols for studying the inflammatory proteomics network in RAW 264.7 cells (a model murine macrophage cell line) with the singular or sequential treatment of LPS and statin. We provide detailed protocols, including a quantitative proteomic analysis by mass spectrometry data, a protein network analysis by bioinformatics, and a validation of target through biochemical methods (e.g., immunocytochemistry, immunoblotting, gene silencing, and real-time PCR).
Keywords: Immunoblotting; Immunocytochemistry; Inflammatory proteomic network; Lipopolysaccharide; Macrophages; Mass spectrometry; Protein-protein interactions; Quantitative proteomics; Statin; Toll-like receptors.