Posttranslational Modifications, Localization, and Protein Interactions of Optineurin, the Product of a Glaucoma Gene
Figure 6
Protein interactions of optineurin.
A. Colocalization of optineurin-GFP foci with Rab8, myosin VI (MyoVI) and transferrin receptor (TfR). RGC5 cells were transfected for 20 h to express optineurin-GFP (OPTN-GFP, in green) and immunostained with anti-Rab8, anti-MyoVI, or anti-TfR.Cy3-labled secondary antibody was used and the Rab8, MyoVI and TfR staining appeared in red (Cy3). Merged images are presented at the bottom panel. Colocalization of optineurin foci with Rab8, MyoVI, and TfR was observed in the perinuclear region in yellow in transfected cells. Those cells that stained positively with anti-Rab8, anti-MyoVI, and anti-TfR in red, but did not display any green fluorescence are non-transfected cells. B. Complex formation of optineurin with Rab8, MyoVI and TfR. RGC5 cells were co-transfected with p-Target-FLAG-OPTN (FLAG-OPTN) and pRab8Q67L-EGFP (Rab8Q67L-GFP), pMyoVI-EGFP (MyoVI-GFP), pTfR-EGFP (TfR-GFP), pRab8Q67L-EGFP + pMyoVI-EGFP, pRab8Q67L-EGFP + pTfR-EGFP, pMyoVI-EGFP+ pTfR-EGFP, or pRab8Q67L-EGFP + pMyoVI-EGFP + pTfR-EGFP. 20 µg of lysates from the transfected cells were subjected to native blue gel electrophoresis and Western blotting using anti-FLAG antibody. Super complexes with molecular sizes larger than 400 kDa were detected. The strongest complex formation was observed between FLAG-OPTN and MyoVI. The complex formation between them was reduced when Rab8Q67L and/or TfR was present. They may compete with each other to interact with optineurin. Size markers are indicated. Arrow denotes the sample loading front.