Assessment of NLRP3 Inflammasome Activation and NLRP3-NEK7 Complex Assembly

The NLRP3 inflammasome is a critical component of innate immunity that activates caspase-1 to induce inflammation in response to a wide spectrum of endogenous and exogenous stimuli. NLRP3 inflammasome activation has been shown by assays for the cleavage of caspase-1 and gasdermin D, the maturation of IL-1β and IL-18, and ASC speck formation in innate immune cells such as macrophages and monocytes. Recently, NEK7 has been revealed as an essential regulator for NLRP3 inflammasome activation by forming high-molecular-weight complexes with NLRP3. Blue native polyacrylamide gel electrophoresis (BN-PAGE) has been used to study multi-protein complexes in many experimental systems. Here, we provide a detailed protocol to detect NLRP3 inflammasome activation and NLRP3-NEK7 complex assembly in mouse macrophages by Western blot and BN-PAGE.