Figure 4.
Time courses of the reactions. A, the reaction was started in a 250-μl mixture under standard conditions, and 15-μl aliquots were withdrawn at the indicated times. B, the reaction was performed as in A except for the use of a 4-fold lower E6AP-E6 concentration. C, the reaction was performed as in A except for the use of a 4-fold lower E2 concentration. D, the reaction was performed as in A except for the use of the UbK48R mutant instead of WT Ub. The reaction products were visualized by Western blotting with the indicated antibodies. Note that because of the presence of cross-reacting material (indicated by the asterisk) in the commercial UbK48R preparation (D), it was not possible to determine whether or not di-Ub was generated in the reaction.