Figure 5. NF-kB factors are present in I. scapularis tick cells and bind to subolesin promoter.

(A) NF-kB-binding activity in ISE6 tick cells nuclear extracts. A DNA-binding ELISA was used with RelA (p65) and RelB antibodies to characterize NF-kB-binding activity in ISE6 tick cells. Nuclear extracts were prepared and assayed from untreated ISE6 tick cells (ISE6), tick cells after incubation with subolesin dsRNA (RNAi SUB) or the unrelated Rs86 dsRNA (RNAi C-), and A. phagocytophilum (A.p) and A. marginale (A.m) infected tick cells. The wild-type consensus NF-kB-binding oligonucleotide was used as a competitor for NF-kB binding in order to monitor the specificity of the assay when compared to non-competing conditions. The mutated consensus oligonucleotide which should have no effect on NF-kB binding was used as negative control (C-) while Raji cells nuclear extracts were used as positive control for NF-kB binding (C+). Results (Ave±S.D.; N = 4) were compared between groups by Student’s T-test (*P≤0.05). (B) Protein sequence alignment of the IPT-NF-kB domain (cd01177) in putative I. scapularis NF-kB factors. Asterisks denote identity to human reference sequence (1A3Q_A). Amino acids conserved in all sequences are shown with (!) at the bottom of the alignment. (C) NF-kB transcription factor binds to subolesin promoter. Representative electrophoretic mobility shift assay showing subolesin promoter-NF-kB interactions. Labeled dsDNA probe corresponding to the subolesin promoter was incubated with p50 and RelA (p65) NF-kB transcription factors. The unlabeled probe used to compete DNA-protein interactions corresponded to the predicted NF-kB-binding site in the subolesin promoter (GTGTCTTTCC). Fast migrating unbound probes are found at the bottom of the gel whereas protein–DNA complexes have slower mobility (arrows).