N-terminally blocked NGN lacking lysines is still ubiquitylated in egg extracts. A, ubiquitylation assays were performed using NGN, Ac2NGN, NGNKO, Ac2NGNKO, and Ac3NGNKO in the presence of His-ubiquitin or ubiquitin. B, ubiquitylation assays were performed with NGN. Reactions were eluted either under non-reducing (Non red) or under reducing/pH 10 conditions (red). C, ubiquitylation assays were performed using NGNKO, Ac2NGNKO, and Ac3NGNKO. Reactions were eluted either under non-reducing or under reducing/pH 10 conditions. Note that NGN and NGN mutant samples were from the same experiment with identical conditions and exposures. Non-modified NGN is marked by *.