HEK293-ILIR cells were incubated without or with IL-Ibeta and HOIP was precipitated from 3 mg cell lysates using immunobilised NEMP (IKK gamma), which captures linear and Lys63-linked ubiquitin chains. Eluted proteins were subjected to SDS-Page before being immunoblotted with anti-HOIP. The results show that NEMO captures HOIP only in IL1-stimulated cells. HOIP was not captured, when NEMO was replaced by the polyubiquitin-binding defective mutants NEMO D311N.
HOIP was immunoprecipitated from HeLa total cell extracts (1 mg) using various amounts of anti-HOIP S174D before being immunoblotted with anti-HOIP (Sigma SAB2102031). The cell supernatants obtained after immunoprecipitations (20 ug) were immunoblotted with anti-HOIP (S174D). 1 ug of anti-HOIP S174D can completely deplete HOIP from 1 mg of cell extract.