Figure 6.
DUB Activity Contributes to Substrate Discrimination In Vitro
(A) The radiolabeled recombinant system was used to analyze mCD4 versus mCD4-M1 ubiquitination in the presence of BSA, HEK293 cell cytosol, or cytosol pretreated with ubiquitin-aldehyde. Note that ubiquitin-aldehyde only partially inhibits DUB activity (Figure S7B). The graph below the autoradiograph depicts the densitometry profiles of the 5 min samples.
(B) The ratio of polyubiquitination acquired on mCD4 divided by that acquired on mCD4-M1 in a parallel reaction is defined as discrimination. This value was determined for three experiments performed as in (A), quantified, and shown in the graph (mean ± SD).
(C) Ubiquitination reactions of mCD4 versus mCD4-M1 in the radiolabeled recombinant system supplemented with the indicated concentrations of the recombinant USP2-CD deubiquitinase. Reaction time was 30 s, although similar results were seen at later time points as well.
(D) Densitometry analysis of the reactions from (C) without or with 8 μM USP2-CD.
(E) Discrimination plotted as a function of USP2-CD concentration derived from (C).
(F) Relative polyubiquitination efficiency of mCD4 and mCD4-M1 as a function of USP2-CD concentration. Values were normalized to the value observed for mCD4 in the absence of USP2-CD.