Meg-01 cells and human platelets contain PPARγ that binds the PPARγ DNA consensus sequence. (A) 15d-PGJ₂ and ciglitazone induce DNA binding of PPARγ protein in MEG-01 cells. After treatment with 15d-PGJ₂ (lane 3) or ciglitazone (lane 4) or DMSO (vehicle control, lane 2), EMSA was performed. Lane 1 was loaded with free probe (no lysate), and lane 5 is nuclear extract from 15d-PGJ₂–treated cells incubated with unlabeled probe (cold competitor) as a control for binding specificity. Lane 6 shows the locations of shifted and supershifted PPARγ (supershift with an anti-PPARγ antibody). Shift assays were repeated 3 times with similar results. (B) EMSA shows that platelets have PPARγ DNA binding activity. Platelet extracts were prepared without any treatment from 3 different pooled platelets as described in “Materials and methods.” Lane 1 shows radioactive-labeled probe. Cell extracts (50 μg) were incubated with 32^P-labeled PPARγ oligonucleotides (lanes 2-4) or cold competitor (unlabeled probe) (lanes 5-7) and run on a 4% nondenaturing gel. Lanes 8 to 10 indicate the locations of supershifted bands with anti-PPARγ antibody. (C) TransAMTM solid-phase PPARγ DNA binding activity measurements show that platelets have some active DNA binding PPARγ without treatment with PPARγ agonist. However, exposure to PPARγ agonist (20 μM 15d-PGJ₂, ciglitazone, rosiglitazone) significantly enhances binding to the PPARγ DNA response element. Assay background in this experiment was 0.02 optical density (OD).