Ubch7

First, E2 was pre-charged in a 5x reaction by incubating 10 μM of UBCH5C or UBCH7 (Boston Biochem) in IVU reaction buffer (30 mM Hepes pH7.4, 100 mM NaCl, 10 mM MgCI²) containing 50 mM ATP, 5 mM DTT, 200 μM FLAG-ubiquitin (Boston Biochem) and 0.2 μM UBE1, for 15 minutes at 37°C. Precharged E2 reaction was diluted to a 1× reaction with substrate (washed bacterial pellet obtained from a 10 cm dish of HeLa cells infected with S.Typhimurium for 4 h or purified LPS (Adipogen)) and E3 ligase (cell lysates or 350 nM recombinant RNF213). The reaction mix was incubated at 37°C for 1 h, after which the reaction was centrifugated at 16,100 g and the bacterial pellet or ubiquitylated LPS was washed twice in IVU reaction buffer containing 4 M urea. The bacterial pellet was lysed in BugBuster, the purified LPS solubilised in BugBuster and further processed for immunoblot analysis as described previously.

A 50 μL reaction mixture for KoiU2 ubiquitination contained 1μM human E1 recombinant protein (Enzo), 5μM UbcH7 (Boston Biochem), 2μM GST-tagged E3 ubiquitin ligase, 5μM T7-KoiU2, 5mM Mg-ATP (Enzo) 2.5μM biotinylated ubiquitin (Enzo) and 1mM DTT in 1× ubiquitination buffer (Enzo). Reactions were assembled at 4°C and initiated at 37°C. Ubiquitination reactions were stopped after one hour by adding 1× Laemlli buffer, after which the samples were boiled for 5 minutes before being loaded onto the SDS-PAGE gel for analysis.