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. 2012 May 25;31(13):3008–3019. doi: 10.1038/emboj.2012.158

Figure 4.

Figure 4

The sumo-dependent degradation of MDC1 following DNA damage is mediated by RNF4 ubiquitination. (A, B) HEK293T cells (A) or MDC1−/− MEFs reconstituted with HA-WT MDC1 or HA-K1840R (B) were treated with MG132 and left unirradiated or irradiated, and the MDC1-RNF4 interaction was examined 4 h later by immunoprecipitation and immunoblot as indicated. (C) HEK293T transfected with Myc-RNF4 or RNF4 ΔSIM mutant were treated as (A) and subjected to immunoprecipitation and immunoblot as indicated. (D) MDC1 ubiquitination was examined in HEK293T cells transfected with control or RNF4 siRNA as Figure 3F. (E) U2OS cells transfected with Ctrl or RNF4 siRNA were treated with IR (5 Gy), and MDC1 levels were examined at indicated time points. (F) Schematic of the combined in vitro sumoylation and ubiquitination assay procedures. (G, H) Bacterially expressed GST-MDC1(aa 1818–2089) WT or K1840R bound to GST-sepharose were subjected to combined in vitro sumoylation and ubiquitination reactions as shown in (F). The GST-MDC1(aa 1818–2089) WT (G) was subjected to in vitro sumoylation reactions in the presence or absence of SUMO1, SUMO2, and SUMO3, and then subjected to in vitro ubiquitination reactions in the presence or absence of FLAG-Ub, RNF4-WT, RNF4-CS, or RNF4 ΔSIM mutant as indicated. GST-MDC1(aa 1818–2089) WT or K1840R (H) was subjected to in vitro sumoylation reactions in the presence of SUMO1, SUMO2, and SUMO3, and then subjected to in vitro ubiquitination reactions in the presence or absence of RNF4 or FLAG-Ub as indicated. The ubiquitination and sumoylation of GST-sepharose-bound protein were analyzed by immunoblot as indicated.