Endogenous mouse ASC is linear ubiquitinated in BMDMs. (a) WT BMDMs were untreated or primed with LPS overnight and stimulated with nigericin for 2 h before IP with a linear ubiquitin-specific antibody in 5M urea denaturing conditions. Immunoprecipitates (IP Lin Ub) and whole cell lysates (WCL) were immunoblotted for ASC and linear ubiquitin (Lin Ub). (b) WT and HOIL-1L^−/− BMDMs were primed with LPS overnight and stimulated with nigericin for 2 h before IP with an ASC-specific antibody and extensive washing in high salt buffer (500 mM NaCl). Immunoprecipitates and WCLs were immunoblotted for ASC and linear ubiquitin. (c) WT BMDMs were primed with LPS overnight and stimulated with nigericin for 2 h before IP with a linear ubiquitin-specific antibody or an equivalent amount of control human antibody in 5M urea denaturing conditions. Immunoprecipitates and WCL were immunoblotted for linear ubiquitin. (d) WCL extracts from lysates in c were immunoblotted for ubiquitin with a linkage nonspecific antibody (Ub). All data are representative of two independent experiments. Protein size markers are indicated in kD.