HEK293-ILIR cells were incubated without or with IL-Ibeta and HOIL1 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-HOIL1. The results show that NEMO captures HOIL1 only in IL1-stimulated cells. HOIL1 was not captured, when NEMO was replaced by the polyubiquitin-binding defective mutants NEMO D311N.
HOIL1 was immunoprecipitated from HeLa total cell extracts (1 mg) using various amounts of anti-HOIL1 S150D before being immunoblotted with anti-HOIL1 (Sigma HP A024185).