Figure 6. NF-κB dependency and subcellular distribution of IL1β-eRNA and IL1β-RBT46(+).

NF-κB dependency in human monocytic THP-1 cells was evaluated using TPCA-1, an inhibitor of IKK2. (a) Cells were incubated with TCPA-1 (10 μM) for 30 min prior to stimulation with LPS (1 μg ml⁻¹) for 60 min. ChIP in combination with qRT–PCR was used to detect NF-κB binding within the promoter regions of IL1β-eRNA, IL1β-RBT46(+), CXCL8 and IL1β. Data were expressed as the fold enrichment of negative control primers, amplifying regions of the genome not transcribed or bound by NF-κB in human monocytes. (b) Cells were incubated with TCPA-1 (indicated concentration) for 30 min prior to stimulation with LPS (1 μg ml⁻¹) for 2 h and the expression of IL1β-eRNA, IL1β-RBT46(+), IL1β and CXCL8 mRNA was measured by qRT–PCR. (c) The subcellular distribution was determined by fractionation into total (T), nuclear (N) or cytoplasmic (C) fractions following exposure to buffer or 1 μg ml⁻¹ LPS for 2 h. Data are the mean±s.e.m. of three independent experiments. Statistical significance was determined using a one-way analysis of variance with Tukey’s multiple comparisons test, where **P<0.01 and ***P<0.001 versus unstimulated vehicle and ^#P<0.05 and ^##P<0.01 versus LPS-stimulated vehicle.