Figure 4.

sLZIP enhances formation of the PPARγ₂ corepressor complex. (a) 293T cells were transfected with GST-PPARγ₂ and Flag-sLZIP, and treated with or without 10 μM Rosi for 24 h. Cell lysates were collected and subjected to a GST pull-down assay using glutathione 4B beads. Cell extracts were analyzed by an immunoprecipitation assay using an anti-Flag antibody. (b) 293T cells were transfected with GST-PPARγ₂ and Flag-sLZIP, and treated with 10 μM Rosi for the indicated time periods. Cell lysates were subjected to a GST pull-down assay. (c) 293T cells were transfected with FABP4-Luc, β-galactosidase, PPARγ₂, and various sLZIP deletion mutants. Following transfection, cells were stimulated with or without 10 μM of Rosi for 24 h. Luciferase activities were normalized to the β-galactosidase activity and presented as the relative activity. All experiments were performed in triplicate and the bar represents the mean±S.D. *P<0.01; **P<0.005. (d) 293T cells were transfected with GST-HDAC3, Myc-PPARγ₂, and Flag-sLZIP, then treated with or without 10 μM Rosi for 18 h. Cell lysates were subjected to a GST pull-down assay using glutathione 4B beads. (e) GST-HDAC3, Myc-PPARγ₂, Flag-sLZIP, and HA-Ub were expressed into 293T cells. Cells were then treated with 10 μM Rosi for 18 h. Cell lysates were subjected to a GST pull-down assay. (f) 293T cells were transfected with GST-PPARγ₂ and Flag-sLZIP, and treated with 10 μM Rosi for 24 h. Cell lysates were subjected to a GST pull-down assay. Protein complex was analyzed on SDS-PAGE followed by western blotting using anti-PGC1α, anti-GST, and anti-Flag antibodies