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. 2012 Feb 6;109(11):4146–4151. doi: 10.1073/pnas.1200448109

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Fig. 5. — WWP2 interacts with and ubiquitinates ARRDC1 to enhance ARMMs release. (A) Deletion of PPXY motifs reduced ARRDC1 ubiquitination and ARMMs release. HEK293T cells were transfected with wild-type or PPXY-deleted (ΔΔPPXY) ARRDC1 fused to mCherry and visualized by confocal microscopy. The corresponding cell lysates and ARMMs were analyzed by Western blotting as indicated. (B) PPXY motifs in ARRDC1 interact with WWP2. HEK293T cells were cotransfected with GFP-tagged WWP2 and HA-tagged wild-type or ΔΔPPXY ARRDC1. Anti-HA immunoprecipitates and whole-cell extracts were examined by Western blotting as indicated. (C) WWP2 expression potentiates ARRDC1 ubiquitination in ARMMs. HEK293T cells were cotransfected with constructs expressing HA-ARRDC1 and WWP2-GFP or the WW domain of WWP2 fused to GFP. Whole-cell extracts and ARMMs from each transfected group were analyzed by immunoblotting as indicated. (D) WWP2 knockdown diminishes ARRDC1 in ARMMs. HEK293T was subjected to transfection with nontargeting (NT) or WWP2-targeting siRNAs. Corresponding whole-cell extracts and ARMMs were analyzed by Western blotting.