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. Author manuscript; available in PMC: 2015 Jul 3.
Published in final edited form as: Mol Cell. 2014 May 29;55(1):15–30. doi: 10.1016/j.molcel.2014.04.030

Figure 6. RNF4 promotes the ubiquitination and degradation of SUMO2/3-modified Atxn1 82Q.

Figure 6

(A and B) Levels of SUMOylated FLAG-Atxn1 82Q (A) and FLAG-nFlucDM-GFP (B), in the absence or presence of RNF4, in HeLa cells treated with or without MG132. d-IP products with similar levels of unmodified proteins and WCL were analyzed.

(C) Levels of SUMOylated FLAG-Atxn1 82Q in HeLa cells that were pre-treated with a control siRNA or a combination of RNF4 siRNAs, analyzed as in (A).

(D and E) Unmodified and SUMO2-modified FLAG-Atxn1 82Q proteins conjugated on M2 beads (+), or control M2 beads (-), were incubated with ubiquitination reaction mixture, in the absence or presence of GST-RNF4. (D) A schematic diagram of the experimental design. (E) WB analysis of FLAG-Atxn1 82Q (left) and GST-RNF4 (right).

(F and G) Localization of Atxn1 82Q-GFP and RNF4 proteins (detected by anti-FLAG antibody) in HeLa. Scale bar: 10 μm.

(H) Effect of the indicated RNF4 proteins on Atxn1 82Q-GFP levels in HeLa cells.

(I) Effect of PML overexpression on Atxn1 82Q-GFP levels in HeLa cells that were pre-treated with control or RNF4 siRNA.

See also Figure S6.