Fig. 4. Overexpression of GNL3L inhibits transcriptional activity of ERR proteins independently of nucleolar distribution. (A1) Estrogen response element (ERE)-specific transcriptional activities were measured in CV-1 cells by the ratio between the ERE-driven firefly luciferase activity and the Renilla-null luciferase activity. ERR elicits a six-fold increase in the ERE-specific transcriptional activity. Coexpression of wild-type GNL3L (WT) leads to a 50% reduction in the ERR-mediated transcriptional activity. This decrease is reversed by a deletion of the ERR-binding I-domain of GNL3L (dI). Coexpression of either the nucleolar form (NoG3l) or the nucleoplasmic form (dBC) of GNL3L suppresses the ERR transcriptional activity more than or to the same extent as the wild-type GNL3L protein. (B1,C1) Using the same approach, we show that this inhibitory activity of GNL3L can also work on (B1) ERR and (C1) ERR with the exception that the dBC mutant has little effect on the ERR-mediated transactivation. Error bars represent the standard error of the mean (± s.e.m.). ^***P<0.0001. (A2,B2,C2) Expression levels of wild-type and mutant GNL3L proteins and ERR proteins in the experimental samples are compared in western blots side-by-side using anti-HA and anti-Myc antibodies, respectively; -tubulin (-Tub) was used as a loading control. (D) GNL3L fails to suppress the estradiol (E2)-induced transcriptional activity of ER on the ERE-driven promoter in the same cell-based reporter system.