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. 2016 Aug 25;7:12638. doi: 10.1038/ncomms12638

Figure 7. USP10 interacts with TOP2α and RNF168 to antagonize RNF168 ubiquitylation of TOP2α and its chromatin binding.

Figure 7

(a,b) HEK293T cells were transfected with Flag-RNF168 and HA-USP10 vectors (a) or Flag-TOP2α and HA-USP10 vectors (b) as indicated. Cells were lysed and IP was performed using anti-Flag and anti-HA antibodies. The resulting precipitates were subjected to IB analysis with the indicated antibodies. WCL, whole-cell lysate. (c) HEK293T cells were transfected with Flag-TOP2α, RNF168, HA-USP10 and Myc-Ub vectors, as indicated. IP using anti-Flag and WCL was subjected to anti-Ub IB analysis to detect TOP2α ubiquitylation. (d) WT MEFs with knockdown of Usp10 (Sh1 and Sh2) and WT controls were examined for the level of Top2α ubiquitylation. Top2α was immunoprecipitated from whole-cell extracts and examined by IB for its ubiquitylation level using anti-Ub. IP using IgG was used as a control. The indicated antibodies were used for IB. (e) HEK293T cells were transfected with Flag-TOP2α with or without RNF168, HA-Ub, USP10 and USP10-C424A as indicated. TOP2α chromatin occupancy in these cells was examined by IB using anti-Flag antibodies and chromatin fractions (Chr). IB analysis of the chromatin fractions is also shown for the indicated antibodies. (f) A simplified model of RNF168-mediated regulation of TOP2α ubiquitylation and decatenation function.