FIG 4.

KSHV RTA induces ID2 degradation through the ubiquitin-proteasome pathway by N-terminal ubiquitination. (A) Domain map of the full-length (WT) ID2. (B) Immunoblot analysis of ID2 WT, D-box point mutant (DBm), or D-box deletion mutant (ΔDB) cotransfected with a vector control or 3xFLAG-tagged RTA. (C) Western blot analysis of WT or lysine less mutant (K-less) of ID2 cotransfected with a vector control or 3xFLAG-RTA for 2 days before being treated with DMSO (vehicle control) or MG132 (40 μM) for 12 h. (D) Immunoblot detection of N- and C-terminal 3xFLAG-tagged ID2 cotransfected with vector control or Myc/His-tagged RTA in 293T cells. (E) Ubiquitin immunoprecipitation was performed using 293T cells cotransfected with Myc/His-RTA (M/H-RTA) and 3xFLAG-ID2 or ID2-3xFLAG. After ubiquitin immunoprecipitation was performed, the samples were subjected to FLAG immunoblot analysis. (F) Immunoblot analysis of 293T cells that were cotransfected with Myc/His-RTA (M/H-RTA) and WT ID2-3xFLAG or its N-terminal truncation mutants. Band intensities of ID2-3xFLAG were quantified relative to tubulin (loading control) and presented as a ratio relative to the vector control of each condition in panel F.