Effect of PPARγ ligands on activation of NF-κB. (A) Nuclear extracts corresponding to 5 μg protein were incubated in the absence of antibody anti-p50, anti-p65, anti–PGC-1, or normal rabbit serum (NRS) in combination with a [³²P]-labeled NF-κB oligonucleotide probe. Arrows indicate migrational location of each nonsupershifted NF-κB–DNA complex. (B) HS-5 cells were transfected with a 3 × NF-κB–binding element–pGL3 promoter luciferase construct. Cells were then pretreated with 1 μM 15-d-PGJ2, 10 μM troglitazone, or 50 μM GW9662 for 2 hours, and then incubated with or without KAS6/1 or ANBL-6 cells for 24 hours. Error bars represent the mean (± SE) from 3 independent experiments. (C) The coactivator PGC-1 coimmunoprecipitates with p50 or p65 in KAS6/1 cell adhesion–induced HS-5 cells. HS-5 cells were treated with or without 1 μM 15-d-PGJ2 or 10 μM troglitazone for 2 hours and then added with KAS6/1 cells for 24 hours before lyses. Western blotting analysis with anti-p65 (top panel), or p50 (middle panel), or anti–PGC-1 (bottom panel) was performed on anti–PGC-1 immunoprecipitates. (D) The coactivator PGC-1 coimmunoprecipitates with PPARγ in HS-5 cells. HS-5 cells were treated with or without 1 μM 15-d-PGJ2 or 10 μM troglitazone for 2 hours and then added with KAS6/1 cells for 24 hours before lyses. Western blotting analysis with either anti-PPARγ (top panel) or anti–PGC-1 (bottom panel) was performed on anti–PGC-1 immunoprecipitates. (E) The above cell extracts were immunoprecipitated with anti-PPARγ. Western blotting analysis was performed with anti-p50 and anti-p65. (F) HS-5 cells were treated with or without 1 μM 15-d-PGJ2 or 10 μM troglitazone for 2 hours and then simulated with KAS6/1 for 1 hour before lyses. Western blotting analysis was performed with anti–phospho-IKKα, anti-IKKα, anti–phospho-IκBα, or anti-IκBα.