Impairment of Protein Trafficking upon Overexpression and Mutation of Optineurin
Figure 7
Self binding of optineurin mutants and their interactions with Rab8 and TfR.
(A) Self binding of wild type optineurin and mutants. RPE cells were co-transfected with pOPTNWT-His + pTarget-FLAG-OPTNWT, pOPTNE50K-His + pTarget-FLAG-OPTNE50K, or pOPTNL157A-His + pTarget-FLAG-OPTNL157A for 16 h. Cell lysates were immunoprecipitated (IP) with polyclonal anti-His. The IP eluates were immunoblotted (IB) with monoclonal anti-FLAG or monoclonal anti-His. The anticipated molecular weight of the proteins detected by both antibodies was 75 kDa. No protein band was detected when rabbit IgG was used for immunoprecipitation (data not shown). (B) Binding of wild type optineurin and mutants with endogenous Rab8. Inducible RGC5 cells were treated with doxycycline for 16 h to express OPTNWT-GFP, OPTNE50K-GFP and OPTNL157A-GFP. Cell lysates were immunoprecipitated (IP) with polyclonal anti-GFP. The IP eluates were immunoblotted (IB) with monoclonal anti-Rab8 or polyclonal anti-GFP. The intensity ratio between the detected 27-kDa-Rab8 band and the 100 kDa-OPTN-GFP band relative to that of the wild type optineurin is presented. No protein band was observed in lysate samples obtained from non-induced cells (data not shown). (C) Binding of wild type optineurin and mutants with endogenous TfR. Inducible stable cell lines were induced by doxycycline to express OPTNWT-GFP, OPTNE50K-GFP or OPTNL157A-GFP. Cell lysates were immunoprecipitated (IP) with a polyclonal anti-GFP antibody. The IP eluates were immunoblotted (IB) with a monoclonal anti-TfR antibody or anti-GFP. The intensity ratio between the 95-kDa TfR band and the 100 kDa-OPTN-GFP band relative to that of the wild type optineurin is presented. No protein band was observed in lysate samples obtained from non-induced cells (data not shown).