Figure 6

15d-PGJ2 inhibits TNFα induced IKKα mediated NF-κB reporter activity. bEND.3 cells were treated with TNFα (50 ng/ml) in the presence or absence of 15d-PGJ2 (10 μM) followed by detection of pIKKα using its specific antibody (Cell Signaling) (A). β actin was used as a control for equal content of protein loaded. bEND.3 cells were transfected with IKKα, NF-κB luciferase and pCMV-β-galactosidase constructs and treated with 15d-PGJ2 (5–20 μM) and TNFα (50 ng/ml). After 4 h of TNFα treatment, cells were processed for luciferase assay as described in 'Material and Methods' (B). Results were calculated as mean ± SD for 3 independent experiments. &&& p < 0.001 compared with control, !!! p < 0.001 compared with TNFα treatment and ### p > 0.001 compared with IKKα. bEND.3 cells were transfected with Gal-p65 or Gal-DBD in the presence or absence of flag-IKKα along with PTL-luciferase and PRL-TK reporter constructs as described in Material and Method. bEND.3 cells were pretreated with 15d-PGJ2 (10 μM) for 30 min followed by TNFα treatment. After 6 h of TNFα treatment (50 ng/ml), cells were processed for luciferase assay and results were normalized with PRL-TK luciferase activity in each sample (C). Total DNA content was normalized with pcDNA3. Results were calculated as mean ± SD for 3 independent experiments.