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. 2016 Sep 9;45(1):92–105. doi: 10.1093/nar/gkw814

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© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 4. — SPOP complex ubiquitinates SETD2 in vivo and in vitro. (A) HEK293 cells were transfected with MYC-SPOP, HA-ubiquitin, FLAG-SETD2-B3 as indicated, followed by MG132 treatment for 12 h. Lysates were immunoprecipitated and Western blotted as indicated. The shifted smear corresponded to poly-ubiquitinated SETD2. (B) FLAG-SETD2-B3 or FLAG-SETD2-B3 (M3) was subjected for ubiquitination assay as in (E). (C) In vivo ubiquitination assay was carried out with HA-tagged full-length SETD2 in the cell. The lysate without SPOP was loaded into the left two lanes with different amounts. (D and E) His-Flag-SPOP, ROC1 and CUL3 were co-expressed by baculovirus and purified as described in the Experimental Procedures. Purified SPOP/CUL/ROC1 complex was incubated with or without E1, E2 and Ub. (F) Purified wild-type SETD2-B3 and the M3 mutant were tested as substrates, respectively, and in vitro ubiqutination assay was carried out as (G).