(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.