FIGURE 6.

RNF4 and the ubiquitination of Rta. A, 293T cells were cotransfected with pCMV-R, pEGFP-RNF4, and pFLAG-Ub (lanes 1–5). At 24 h after transfection cells were treated with 5 μm MG132 for another 12 h. Proteins in the lysate were immunoprecipitated (IP) using anti-FLAG antibody. Proteins bound to FLAG-M2 beads were analyzed by immunoblot analysis (IB) using anti-Rta and anti-GFP antibodies. Similarly, 293T cells were cotransfected with plasmids that express FLAG-Rta, GFP-RNF4, and FLAG-Ub (lanes 6–10). At 24 h after transfection, cells were treated with 5 μm MG132 for another 12 h. Proteins in the lysate were immunoprecipitated using anti-FLAG antibody. Proteins bound to FLAG-M2 beads were analyzed by immunoblot analysis using anti-HA, anti-Rta, and anti-GFP antibodies. B, shown is the effect of knockdown of RNF4 on the ubiquitination of Rta. 293T cells were cotransfected with plasmids that express FLAG-Ub, Rta, and RNF4 shRNA or control shRNA (Ct-shRNA) and then treated with 5 μm MG132 for 12 h. Ubiquitinated Rta was immunoprecipitated using anti-FLAG antibody and detected by immunoblotting using anti-Rta antibody. C, 293T cells were cotransfected with pHA-Ub, pCMV-R, and pFLAG-RNF4 or pFLAG-RNF4-CS1. At 24 h after transfection, cells were treated with 5 μm MG132 for another 12 h. Proteins in the cell lysate were immunoprecipitated using anti-HA antibody. Immunoprecipitated proteins were detected by immunoblotting with anti-Rta antibody. D, a similar experiment as described in C was conducted except that pFLAG-RNF4-CS1 was replaced with pFLAG-RNF4-mSIM. E, interaction of Rta with RNF4 mutants is shown. 293T cells were cotransfected with pEGFP-Rta and plasmids that express FLAG-RNF4, FLAG-RNF4-CS1, or FLAG-RNF4-mSIM. A coimmunoprecipitation assay was performed using anti-FLAG antibody, and immunoprecipitated proteins were detected by immunoblotting with anti-GFP antibody (lanes 6–10). Asterisks indicate bands detected nonspecifically. Ub-Rta, ubiquitinated Rta.