FIG 2.

KSHV RTA induces the polyubiquitination and degradation of ID2 through the ubiquitin-proteasome pathway. (A) Immunoblot detection of 3xFLAG-RTA and ID2 in Dox-induced iBJAB-3xFLAG-RTA cells. (B) RT-qPCR analysis of ID2 transcript abundance in iBJAB-3xFLAG-RTA cells. t tests were performed between 0 h postinduction (hpi) and 24 or 48 hpi (ns, not significant). (C) Immunoblot analysis of lenti-GFP and lenti-ID2-transduced iSLK cells expressing Dox-inducible RTA. (D) Western blot analysis of HeLa cells cotransfected with ID2 and either vector control or an increasing amount of 3xFLAG-RTA. (E) Immunoblot detection of 3xFLAG-RTA and ID2 in 293T cells that were cotransfected with ID2 and either vector control or increasing amounts of 3xFLAG-RTA. (F) RT-qPCR analysis of ID2 in the experiment performed in panel E when control vector or the largest amount of RTA was cotransfected with ID2. (G) Western blot analysis of 293T cells that were cotransfected with ID2 and vector or 3xFLAG-RTA, and treated with DMSO (control) or 40 μM MG132. (H) FLAG immunoprecipitation was performed using 293T cells that were cotransfected with ID2-3xFLAG and either a vector control or Myc/His-tagged RTA (M/H-RTA). FLAG IP was subjected to ubiquitin Western blotting. (I) 293T cells were cotransfected with HA-ubiquitin, ID2-3xFLAG, and Myc/His-RTA (M/H-RTA). After FLAG immunoprecipitation (IP) was performed, the IP samples were subjected to immunoblot analysis.