Figure 7
DMF can inhibit E2 conjugating enzymes in vitro.
(A) Reaction mechanism of DMF and cysteine. (B) Ubc13 or UbcH7 was incubated in the presence or absence of DMF for 30 minutes. E2 loading reactions for Ubc13 and UbcH7 in the presence of ubiquitin, Ube1 and Mg-ATP were carried out in the presence of increasing concentrations of DMF as described in the methods. Ubiquitin loading was resolved on 4-12% polyacrylamide gels. Example gels are shown on the left and the quantification of 3 separate reactions shown on the right. Error bars represent standard deviation. For DMF treated conditions, a p value of less than 0.001 (Students t-test) is indicated by ***. (C) Ubc13 or UbcH7 was incubated in the presence or absence of 50 μM DMF for 4 h. The molecular mass was then determined by MALDI-TOF mass spectrometry. Spectra show intensity vs. m/z and inserts show an expansion of the 2+ peak. (D) The indicated E2 conjugating enzymes were incubated with either 25 or 100 μM DMF for 30 minutes and E2 loading assays carried out as described in the methods. The % E2 loading relative to the 0 μM condition for each E2 tested is shown.
