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. Author manuscript; available in PMC: 2017 Jun 16.
Published in final edited form as: Mol Cell. 2016 May 26;62(6):958–966. doi: 10.1016/j.molcel.2016.04.022

Figure 3. SETDB1-K867Ub1 is protected from deubiquitination.

Figure 3

(A) Flag-SETDB1-(Δ528–584) or (Δ1210–1291) was co-expressed together with UBE2E1 and 13 DUBs in 293T cells while WCE derived from transfected cells was analyzed by western blot using indicated antibodies.

(B) IP-purified Flag-SETDB1-(Δ528–584) and (Δ1210–1291) deletions were ubiquitinated in vitro by UBE2E1 and then incubated with recombinant USP2A [2A-wt or C276A (DN)] or USP17 [17-wt or C89S (DN)] for in vitro deubiquitination. Products were analyzed by western blot using Flag antibody.

(C) Flag-SETDB1-wt, (Δ528–584) or (Δ1210–1291) was expressed at endogenous level in SETDB1-KD 293T cells together with UBE2E1 and USP2A or USP17 (wt or DN). WCE derived from different transfected cells was analyzed by western blot using indicated antibodies.

(D) IP-purified Flag-SETDB1- wt, (Δ528–584) or (Δ1210–1291) was in vitro ubiquitinated by UBE2E1 and then incubated with recombinant USP2A or USP17 for in vitro deubiquitination. Products were analyzed by anti-Flag western blot.

(E) Flag-SETDB1 was co-expressed with USP17 in 293T cells while minimally ubiquitinated Flag-SETDB1 was IP-purified and further ubiquitinated in vitro with Ub-wt or Ub-I44A by UBE2E1. Products were then purified and incubated with recombinant USP17 (wt or DN) for in vitro deubiquitination followed by western blot analysis.

(F) Flag-SETDB1 was co-expressed with HA-Ub-wt or I44A in 293T cells and analyzed by IP-western using indicated antibodies.

See also Figure S3.