FIGURE 2.

ARRDC3 is ubiquitinated and interacts with the ESCRT-0 complex. A, co-immunoprecipitation of transiently transfected pEGFP-ARRDC3 or pEGFP-ARRDC3-AASA/AALA with FLAG-STAM-1 in HEK293 cells. B, representative immunofluorescence images and corresponding line scan analysis demonstrating the co-localization of expressed pEGFP-ARRDC3 and endogenous STAM on endosomes in HEK293 cells. Endosomes were enlarged by transient transfection of Rab5-Q79L. Scale bars of the insets, 1 μm. C, top, representative immunofluorescence images are shown to demonstrate the localization of WT and mutant pEGFP-ARRDC3 in HEK293 cells. Middle and bottom, same cells were co-stained for endogenous STAM to evaluate the co-localization with the ESCRT-0 complex. D, ubiquitination of WT and mutant GFP-ARRDC3 was evaluated by immunoprecipitating ARRDC3 followed by detection of endogenous ubiquitin by Western blotting. All experiments are done at least three times. Scale bars, 5 μm. WCL, whole cell lysate; IB, immunoblot.