FIGURE 6.

NF-κB-sensitive transcription of Orai1 and STIM1. A, analysis of the DNA sequence of 11p15.5 in the region adjacent to the STIM1 transcriptional start site (S1) and of 12q24.31 in the region adjacent to the Orai1 transcriptional start site (O1 and O2) revealed putative κB-like binding sites (italic, underlined) is shown. For ChIP experiments DNA was amplified by PCR and subcloned into pGL3-vector for luciferase reporter assay using the primers depicted in bold. B, a luciferase reporter assay was applied to study the ability of NF-κB to activate the target promoters of STIM1 and Orai1. HEK293 cells were transfected with luciferase reporter plasmids containing the putative κB-binding sites and additionally transfected with p65 or control plasmid. The results are presented as -fold increase in the activity of luciferase activity (±S.E., n = 11–21) compared with luciferase activity transfected with the control plasmid. * (p < 0.05) indicates statistically significant differences (ANOVA). C, binding of NF-κB to DNA was explored utilizing ChIP of genomic DNA extracted from HEK293 cells transfected with p65 or a control plasmid. Protein-chromatin complexes were immunoprecipitated with anti-p65 or anti-rabbit IgG. The input served as a positive control, IgG as a negative control. The experiment was carried out five times.