Figure 5.

Reconstitution of conjugation of h-N-myc from purified conjugating enzymes. h-N-myc translated in vitro in wheat germ extract that lacks E6-AP was incubated for 10 min at room temperature in the presence of 10 mM N-ethylmaleimide to inactivate the endogenous E1 and E2 enzymes. DTT was added to a final concentration of 6 mM to neutralize the N-ethylmaleimide. Conjugation reactions were carried out in the presence of purified E1, E2-F1, and either GST-E6-AP or baculovirus-expressed E6-AP and GST-E6-16 as indicated.