Figure 9

LG268 and rosiglitazone induce SUMOylation of their respective receptors and the unliganded partner. LG268 and rosiglitazone treatment increases SUMOylation of (a) peroxisome proliferator-activated receptor-gamma (PPARγ) and (b) retinoid X receptor (RXR). SW-1353 cells were treated with 1 hour of 50 nM LG268, 50 nM rosiglitazone, or both for pretreatment and then 1 hour of 1 ng/mL interleukin-1-beta (IL-1β) for stimulation. Cells were harvested in 1× radioimmunoprecipitation assay lysis buffer, and total protein was quantified. Two thousand micrograms of total cell protein and 4 μg of anti-SUMO1 antibody were used in each immunoprecipitation reaction. Immunoprecipitated proteins were resolved using PAGE; after transfer, Western blotting was performed with anti-PPARγ or anti-RXR antibody at a dilution of 1:2,000. Bands were visualized using enhanced chemiluminescence and overnight autorad exposure. IgG bands are displayed as a loading control. (a) Single arrowhead denotes high-molecular weight/2 × SUMO1-PPARγ band. (b) Double arrowhead denotes high-molecular weight/2 × SUMO1-RXR band; single arrowhead denotes low-molecular weight/1 × SUMO1-RXR band. Lane reference numbers 1P to 8P and 1R to 8R are displayed to facilitate description in the text. IP, immunoprecipitation; LG268, LG100268; Rosi, rosiglitazone; SUMO, small ubiquitin-like modifier; WB, Western blotting.