Figure 2

Optineurin is localized to the recycling endosomes. (A) Endogenous optineurin is localized to recycling endosomes in HeLa cells. HeLa cells were stained sequentially for endogenous optineurin and TfR (upper panel) or were transfected with GFP-Rab11 and stained for endogenous optineurin (lower panel). Arrows indicate vesicles showing co-localization. (B) HeLa cells were pulsed with Alexa-546 conjugated transferrin for 30 min, chased with unlabeled holo-transferrin for 20 min and stained for endogenous optineurin. Arrows indicate vesicles showing colocalization. (C) RGC-5 cells were stained for endogenous optineurin and TfR. (D) E50K mutant optineurin forms large vesicles and co-localizes with TfR. HeLa cells were transfected with either E50K mutant or wild-type optineurin expressing plasmids lacking a tag and stained sequentially for optineurin and TfR. E50K mutant optineurin forms large vesicular structures that co-localize strongly with TfR indicated by yellow colour in the merged image. Right panels show enlarged image of boxed area in merged panels. Bar; 10 μm.