FIG. 3.
Ataxin-3 can interact with multiubiquitin chains. (A). GST, GST-ataxin-3Q20, and GST-ataxin-3Q79 were bound to glutathione Sepharose 4B. GST-S5a was purified on glutathione agarose (Affiniti). One and five micrograms of an equimolar mixture of (Ub)2-4 (Affiniti) were incubated with the beads, and bound proteins were resolved on an SDS-10% polyacrylamide gel, transferred to nitrocellulose membranes, and detected with ubiquitin-specific antibodies. Lanes: 2 and 3, GST; 4 and 5, GST-ataxin-3Q20; 6 and 7, GST-S5a; 8 and 9, GST-ataxin-3Q79. One microgram of (Ub)2-4 was added to lanes 1, 2, 4, 6, and 8, while 5 μg was added to lanes 3, 5, 7, and 9. Ponceau staining shows that the protein levels were similar. (B) UIMs of ataxin-3 are necessary for binding to ubiquitin chains. GST-S5a was purified on glutathione agarose (Affiniti). GST, full-length GST-ataxin-3Q20 (Q20), full-length GST-ataxin-3Q79 (Q79), and GST-ataxin-3 truncation proteins were purified on glutathione Sepharose, and equal amounts were incubated with 1 μg of (Ub)4 (Affiniti). The interactions were determined by immunoblotting with ubiquitin-specific antibodies (Sigma). Lane 1, GST-S5a; lane 2, GST; lane 3, GST-ataxin-3Q20; lane 4, GST-ataxin-3Q79; lane 5, GST-ataxin-31-150; lane 6, GST-ataxin-31-200; lane 7, GST-ataxin-31-243 (UIM1); lane 8, GST-ataxin-31-263 (UIM2); lane 9, GST-ataxin-31-354 (UIM3); lane 10, 100 ng of (Ub)4; lane 11, 1 μg of (Ub)4. Ponceau staining shows that equal amounts of GST proteins were present.