Blocking of NF-κB and AP-1 pathways in HRS cells partially inhibited LTα production. (A) Western blot analysis and band density quantification showed reduced nuclear p65 expression in Bay11-7085–treated KM-H2 cells in a dose-dependent manner. (B) Treatment of KM-H2 cells with 3 different doses of Bay11-7085 as indicated resulted in reduced LTα production compared with untreated and DMSO-treated controls. (C-D) Western blot analysis and band density quantification showed reduction of phosphorylated and total c-Jun in SP600125-treated KMH2 cells. (E) Expression of phosphorylated c-Fos in these cells was unaffected. (F) KM-H2 cells treated with SP600125 showed reduced LTα production, with the most significant reduction observed when 80 and 100 µM of the inhibitor was used. Western blot shown is representative data of 3 independent experiments for A, C, and D and 2 independent experiments for E. The TATA box served as a nuclear protein loading control. β-Actin served as cytoplasmic protein loading control. Quantification of band density was carried out using ImageJ 1.48 software (National Institutes of Health). Density of the protein band for each condition was first normalized to respective loading control and then the fold change over untreated (UT) control cells was determined. Values shown are the mean ± standard deviation from 2 independent experiments for B and E and mean ± SEM from 3 different experiments for A, C, D, and F. *Statistical significance at P ≤ .05 compared with UT KM-H2 cells.