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. 2017 Jan 6;12(1):e0169587. doi: 10.1371/journal.pone.0169587

Fig 2. DUB3 interacts with ITCH and mediates its stability.

Fig 2

(A) Immunoblots showing the effect of DUB3 expression on ITCH. HEK293T cells were transfected with a vector expressing Flag-DUB3, its C89S DUB3 or a control vector. Blots were probed with anti-Flag, anti-ITCH, anti-NEDD4 and anti-SMURF1 antibodies. Anti-Actin was used to control for loading. (B) Immunoblots showing the effect of DUB3 siRNAs on ITCH. HEK293T cells were transfected with independent siRNAs against DUB3 or a scrambled siRNA. Blots were probed with antibodies against ITCH or Actin for loading control. (C) Immunoprecipitation assays showing interaction between DUB3 and ITCH. HEK293T cells were transfected to express Flag-tagged DUB3 and Myc-tagged ITCH as indicated. Transfected cells were treated with MG132 5μM overnight before being harvested for immunoprecipitation with anti-Flag or anti-Myc-conjugated beads. Blots were probed with anti- Flag to detect DUB3 or anti-Myc to detect ITCH. (D) Ubiquitylation assay showing the effect of DUB3 on ITCH ubiquitylation. HEK293T cells were co-transfected with a vector expressing Myc-ITCH and a vector expressing Flag-tagged DUB3, Flag-tagged DUB3 C89S or a control vector. Transfected cells were treated with MG132 5μM overnight before being harvested for immunoprecipitation with anti-Myc-or isotype IgG-conjugated beads in PLC buffer freshly supplemented with 10mM of NEM. Immunoblots were probed with antibodies against HA, Flag and Myc.