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. 2013 Aug 1;154(3):609–622. doi: 10.1016/j.cell.2013.06.038

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© 2013 The Authors

Open Access under CC BY 3.0 license

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Figure S7 — DUBs Enhance Substrate Discrimination In Vitro, Related to Figure 6

(A) Radiolabeled mCD4 isolated from in vitro translation reactions was coreconstituted with recombinant Vpu or Vpu-M1 into liposomes and subjected to ubiquitination in the presence of BSA, HEK293 cell cytosol, or cytosol pretreated with ubiquitin-aldehyde. The reaction at different time points was stopped and the analyzed for ubiquitinated mCD4 via pulldowns of His6-tagged ubiquitin (Ub PDs). Total mCD4 in the reaction is shown in the bottom panel. The graphs below the autoradiographs depicts the densitometry profiles of the 5 min samples.

(B) Proteoliposomes containing ubiquitinated mCD4 or mCD4-M1 generated as in Figure S4B were isolated and incubated with HEK293 cytosol, or cytosol pretreated with ubiquitin-aldehyde. Samples were removed at different time points and analyzed for ubiquitinated products by pulldowns and autoradiography. Note that deubiquitination activity is reduced, but not completely eliminated, by ubiquitin-aldehyde pretreatment.

(C) An experiment as in B except that the cytosol was supplemented with the SCF complex and E1/E2 enzymes. Note that initially, deubiquitination is observed, but over time, this activity is counteracted by the SCF.