<p>(A) HeLa cells were induced with IFN-β for 24 h. The cells were lysed in urea buffer without reducing agent. Cellular lysates were equally aliquoted and 2-ME was added (loading to SDS-PAGE from left to right: 500 mM, 100 mM, 50 mM, 20 mM, 10 mM, 5 mM, 1 mM 2-ME, empty lane, 0 mM 2-ME) and blotted for MxA and hGBP1. (B) HeLa cells were transiently transfected with pCMV2b-Flag-MxA and the components of the ISG15 conjugation machinery as indicated in the figure. 24 h post-transfection, the cells were induced with IFN-β24 post-induction the cells were collected and lysed without 2-ME. Anti-FLAG immunoprecipitations were performed without 2-ME. Eluates were equally split and treated with or without 2-ME before SDS-PAGE (C) HeLa cells were transiently transfected with pCMVb-HA-MxA and components of the ISG15 conjugation machinery as indicated in the figure. 24 h post-transfection, the cells were collected and lysed without 2-ME. Anti-HA immunoprecipitations were performed. Eluates were equally split and treated with or without 2-ME before SDS-PAGE. (D) HeLa cells were transiently transfected with either pCMVb-MRGS-His-ISG15 or pCDNA4/TO/N-MRGS-His-SUMO2. 24 h post-transfection, the cells were induced with IFN-β (1,000 units/ml). Purifications of ISG15 or SUMO2 modified proteins were carried out under denaturating conditions without 2-ME. Eluates were equally split and treated with or without 2-ME before SDS-PAGE.</p
