IKK kinase assay

MCF10A-ras cells were cultured in 100-mm dishes in the absence or presence of 15d-PGJ₂. When necessary, DTT (500 μM) was added. The protein was isolated as described above for the Western blot analysis. The lysed cell extract (200 μg) was precleared using normal mouse immunoglobulin G and protein G agarose beads and subjected to immunoprecipitation by using anti-IKKβ antibody. The resulting immunocomplex was pulled down by mixing with protein G agarose beads. The immunoprecipitates were suspended in 50 μL of a reaction mixture containing 1X kinase buffer (Cell Signaling Technology, Beverly, MA, USA), 1 μg glutathione S-transferase-IκBα as a substrate and 10 μCi of [γ-³²P]ATP and incubated at 30°C for 45 minutes. The kinase reaction was terminated by adding SDS loading dye, boiled for 5 minutes at 99°C, vortexed and centrifuged at 5,000 rpm for 2 minutes. The supernatant was separated by 12% SDS-polyacrylamide gel. The gel was stained with Commassie Brilliant Blue G 250 and treated with destaining solution (glacial acetic acid:methanol:distilled water, 1 : 4 : 5, v/v). The destained gel was dried at 80°C for 1 hour and exposed to X-ray film to detect the phosphorylated glutathione S-transferase-IκBα in the radiogram. Mouse immunoglobulin G heavy chain band that appeared on the destained gel was used as the loading control to ensure the equal lane loading.