FIGURE 7.

SUMO-dependent ubiquitination of Rta by RNF4. A, 293T cells were cotransfected with pFLAG-Ub, pEGFP-RNF4, and pHA-Rta (lanes 2–4), pHA-4K-R (lanes 5 and 6), or pHA-3K-R (lanes 7 and 8). 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 were then detected by immunoblotting (IB) using anti-Rta and anti-GFP antibodies. B, interaction of RNF4 with Rta is shown. 293T cells were cotransfected with pFLAG-RNF4 and plasmids that express HA-Rta, HA-3K-R, or HA-4K-R. A coimmunoprecipitation assay was performed using anti-FLAG antibody, and immunoprecipitated proteins were detected by immunoblotting using anti-HA antibody. C, P3HR1 cells were transfected with plasmids pEGFP-RNF4 (a–r), and then cells were treated with 5 μm MG132 or DMSO for 12 h after lytic induction by TPA and sodium butyrate. Cells were incubated with anti-Rta monoclonal antibody and anti-SUMO-2 polyclonal antibody. DAPI staining revealed the nucleus. Thereafter, cells were observed under a confocal laser-scanning microscope. d, h, m, and r are merged images.