Figure 3
The UBD is required for localization of optineurin to RE. (A) HeLa cells were transfected with GFP-tagged wild type optineurin or its mutants and stained for TfR. Ubiquitin binding defective D474N and E50K-D474N mutants are not recruited to RE as indicated by a lack of colocalization with TfR. Note the absence of large vesicular structures in E50K-D474N mutant. Bar; 10 μm. (B) The number of vesicles in cells expressing GFP tagged wild-type and mutant optineurin. (C) Western blot showing expression of GFP tagged optineurin and its mutants, probed with optineurin antibody. (D) E50K-D474N mutant does not bind ubiquitin. GST-ubiquitin or GST alone bound to glutathione agarose beads were incubated with lysates of HeLa cells transfected with wild type optineurin or its mutants. The bound proteins were eluted and immunoblotted with anti-HA antibodies. WCL, 5% whole cell lysate.