Figure 1

Optineurin is required for the trafficking of TfR. (A) Schematic representation of optineurin showing domain organisation, key mutations and binding sites of interacting proteins. UBD- Ubiquitin Binding Domain (424-511aa), CC-coiled coil domain, ZF- zinc finger domain. (B) HeLa cells were infected with adenoviruses expressing shRNAs (Ad shOPTN1 and Ad shOPTN2) against optineurin for 72 hrs. Western blots show levels of endogenous optineurin, TfR and tubulin. (C) HeLa cells were infected with adenoviruses expressing shRNAs for 72 hrs, serum starved for 2 hrs and then incubated with Alexa-546 conjugated transferrin for 15 min, stained for transferrin receptor and examined by confocal microscopy. (D) Expression of shRNA resistant mutant of optineurin in HeLa cells. (E) HeLa cells infected with adenoviruses expressing shRNA (Ad shOPTN2) were transfected with a shRNA resistant mutant of optineurin and were incubated with Alexa 546-Tfn for 15 min. Expression of shRNA resistant mutant (right panels, shown in green) rescues the effect of shRNA. Bar; 10 μm.