Fig. 1.

Inflammatory mediators increase SUMOylation of RXRα. (A) HuH-7 cells were transfected with F-RXRα, UBC9 and c-myc (empty vector; Vect.), SUMO1, or SUMO2. Cells were treated for 4 hours with vehicle or LPS, 1 mg/ml, or IL-1β, 10 ng/ml, for 30 minutes with TNFα, 10 ng/ml, and harvested and immunoblotted with anti-FLAG. (B) Densitometric analysis of blots treated as per A and including a minimum of three separate experiments, where the SUMO-F-RXRα band intensity is plotted relative to the F-RXRα band. Vehicle-treated samples are normalized to 1.0, data are presented as mean + S.E. and *P < 0.05, relative to the vehicle-treated control. Empty vector samples are not graphed because there is no detectable SUMO–F-RXRα band. (C) HuH-7 cells were transfected with F-RXRα and SUMO1 and pretreated with vehicle (Veh) or SUMO inhibitors 100 μM AA or 25 μM GA for 4 hours prior to treatment with vehicle or TNFα as in A. (D) HuH-7 cells transfected and treated with His-tagged RXRα and myc-tagged SUMO1 as in A then His-purified and immunoblotted with anti–c-myc antibody. Blot shows three different samples for both vehicle- and TNFα-treated cells. Note: all images are representative blots from a minimum of three experiments. Arrow denotes F-RXRα, and arrowhead denotes Su–F-RXRα. NSP, nonspecific.