Covalently bonded oligomers of optineurin are induced by oxidative stress.

A. NIH3T3 cells transfected with FLAG-OPTN were incubated without or with 25 mM H₂O₂ for 20 min and then analyzed by SDS-PAGE and western blotting with anti-FLAG antibody. A protein complex (right lane, arrow) was observed in the H₂O₂-treated sample. Other high molecular weight bands (asterisk) were seen at low density in both untreated and H₂O₂-treated samples. The optineurin (OPTN) monomer was observed at the expected position (arrowhead) B. After standard transfection into NIH3T3 or HeLaS3 cells and treatment without or with FLAG-OPTN and H₂O₂, lysates were immunoprecipitated with anti-OPTN antibody for detection of endogenous OPTN (left panel), or anti-FLAG antibody for detection of transfected OPTN. The H₂O₂-induced protein band was detected by anti-OPTN antibody in all cases (arrow). Both endogenous and transfected OPTN monomer band was observed at an expected position (arrowhead). Another band was seen above the monomer in the lane of transfected OPTN, but it was not analyzed in this study because it was not present in the case of endogenous OPTN. C. NIH3T3 cells expressing GFP-OPTN and FLAG-OPTN, or both, were treated without or with H₂O₂. Cell lysates were immunoprecipitated with anti-FLAG antibody and analyzed by western blotting with antibodies against FLAG (lanes 1–6) or GFP (lanes 7–12). The H₂O₂-induced bands were detected in co-transfected and FLAG-transfected samples (lanes 2, 4, and 8, arrows). OPTN monomer bands were shown as arrowheads. IB: immunoblotting; IP: immunoprecipitation.