Figure 11From: Regulation of endocytic trafficking of transferrin receptor by optineurin and its impairment by a glaucoma-associated mutantThe E50K mutant is altered in its interaction with Rab8. (A) E50K mutant shows enhanced interaction with activated Rab8. HeLa cells were infected with adenoviruses expressing HA-E50K or HA-optineurin and then transfected with Myc-Q67L-Rab8 mutant. After 36 hrs of infection, lysates were prepared and immunoprecipitated with anti-Myc or control antibody and subjected to Western blotting with TfR, HA and anti-Myc antibodies. WCL, 5% whole cell lysate. (B) Co-localization of E50K mutant with Q67L-Rab8 and TfR. HeLa cells were cotransfected with GFP-tagged Q67L-Rab8 along with HA- tagged E50K mutant or wild type optineurin. Cells were stained sequentially with antibodies against HA-tag (red) and transferrin receptor (blue). Pairwise colocalizations are shown as indicated. Bar; 10 μm. (C) E50K mutant affects velocity of Rab8 vesicles. HeLa cells were co-transfected with HA-E50K or HA-optineurin and GFP-Rab8 and the movement of Rab8 vesicles was tracked to measure their velocities. In cells co-expressing E50K velocity of Rab8 vesicles is reduced compared to those expressing wild type optineurin or Rab8 alone.Back to article page