Figure 5.

USP4 Counteracts Its Own Ubiquitylation

(A) GFP-USP4 WT but not CD immunoprecipitations from U2OS cell extracts retrieved CtIP and RAD50 in presence or absence of EtBr (50 μg/ml; see Figure S5A for inputs).

(B) HA-ubiquitin (HA-Ub) immunoprecipitations (in presence of 1 M NaCl) retrieved GFP-USP4 CD but no detectable GFP-USP4 WT from U2OS cell extracts.

(C) GFP-immunoprecipitations from U2OS cell extracts that expressed HA-ubiquitin and GFP, GFP-USP4 WT, or CD, followed by western blot analysis with an HA antibody retrieved HA-ubiquitylated forms of GFP-USP4 CD and to a lesser extent GFP-USP4 WT (130 or 250 indicated respective protein sizes in kDa).

(D) HA-Ub immunoprecipitations from U2OS cell extracts that were processed in absence of β-mercaptoethanol (βME) retrieved modified forms of GFP-USP4 CD that were not visible in presence of βME (exp., exposure; GFP cells were the control).

(E) GFP immunoprecipitations from U2OS cell extracts that were processed in absence of βME, retrieved modified forms of GFP-USP4 CD, and to a lesser extent GFP-USP4 WT, which were not visible in presence of βME.

(F) HA-Ub immunoprecipitations (followed by western blotting analysis) from U2OS cell extracts expressing various GFP-fused USP4 derivatives retrieved GFP-USP4 CD and CD-C799A, but less efficiently the other zinc-binding motif cysteine mutants (CD-C461A, CD-C464A, and CD-C802A; IgG, IgG heavy chain). All samples were run on the same SDS poly-acrylamide gel. See also Figure S5 and Table S4 (describes all ubiquitin sites identified by tandem mass spectrometry on USP4 WT and CD).