Figure 4.

DHX15 binds poly I:C via HELICc domain. (A) Immunoblot (IB) of indicated proteins precipitated with NeutAvidin beads from whole-cell lysates of biotin-labeled poly I:C or biotin-labeled poly dG:dC-stimulated D2SC cells. Whole-cell lysate of unstimulated D2SC cells (Input) served as a control. (B) Immunoblot using anti-Flag antibody of pulldown competition assays in which 0.5, 5, or 50 μg/ml of poly I:C or poly dG:dC was added to a mixture of Flag-DHX15 protein plus biotinylated poly I:C, followed by addition of NA-beads. (C) Immunoblot using anti-HA antibody of pulldown assays in which HA-tagged serial truncations of DHX15 were individually incubated with biotinylated poly I:C, followed by addition of NA-beads. Top, schematic representations of full-length and serial truncations of DHX15. a, DHX15 full size; b, DHX15 deleted C-terminal HA2 and DUF domains; c, N-terminus of DHX15 with DEXDc domain; d, C-terminus of DHX15 with DUF domain. DEXDc: DEAD-like helicases superfamily domain; HELICc: helicase C-terminal domain. HA2: Helicase associated domain; DUF: domain of unknown function. Numbers denote amino acid residues. (D) Anti-HA antibody blotting (IB HA) showing the expression of HA fusions in D2SC cells transfected with empty vector (−) or expression vectors for wild type DHX15 or its mutants individually. GAPDH blots are shown as loading controls. (E) Activation of the NF-κB promoter in D2SC cells transfected with an NF-κB luciferase reporter (NF-κB-Luc; 100 ng) plus increasing concentrations (50, 100 or 200 ng) of expression vectors for wild type DHX15 or its mutants individually. At 24 h posttransfection, cells were stimulated for 6 h with 5.0 μg/ml poly I:C delivered by Lipofectamine^™ 2000 and then harvested. Results are presented relative to those of cells transfected with empty vector alone (−). Data are representative of three independent experiments (mean and s.d. in e).