Fig. 1.

Ubiquitination and degradation of ligand-free LXRα. A, LXRα-expressing HEK293 cells were pretreated with MG132 (20 μm) for 30 min and harvested at 1, 3, and 6 h after cycloheximide treatment (10 μg/ml). After isolating total lysates, Western blottings were preformed. B, The ubiquitination of LXRα was examined by in vivo ubiquitination assays, as described in Materials and Methods. Briefly, LXRα and/or HA-tagged ubiquitin expression vector was cotransfected into HEK293 cells, and the cells were incubated with MG132 (20 μm) for 3 h before harvesting. After isolating total cell lysates, coimmunoprecipitation and Western blotting analysis were performed with indicated antibodies. All the experiments were independently repeated at least three times, and representative results were shown. CHX, Cycloheximide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IB, immunoblotting; IP, immunoprecipitation.