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Figure 11 | BMC Cell Biology

Figure 11

From: Regulation of endocytic trafficking of transferrin receptor by optineurin and its impairment by a glaucoma-associated mutant

Figure 11

The 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.

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