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. 2019 Sep 18;10:2202. doi: 10.3389/fimmu.2019.02202

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Copyright © 2019 Ruan, Cao, Ling, Li, Wu, Peng, Wang, Jia, Chen, Xu and Yuan.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Isolation of poly(I:C) binding proteins from amphioxus intestinal cells. (A) Silver stained poly(I:C)-associated proteins from amphioxus intestinal cells purified with the poly(I:C) agarose or control poly(C) agarose. Proteins identified by LC-mass spectrometry. Two poly(I:C) lines indicated samples from two separated experiments. (B) The comparison of protein architectures between Drosophila, amphioxus and human DDX23, DHX9, and DHX15. All these helicases have highly conserved DEXDc (DEAD-like helicases superfamily) and HELICc (helicase superfamily c-terminal) domains. (C) Pull-down assays were performed by incubating purified myc-bbeDHX15 or myc-bbeDDX23 with the poly(I:C) or poly(C) agarose. Bound proteins were analyzed by immunoblotting with anti-myc. (D) The mixture of myc-bbeDDX23 or myc-bbeDHX15 and poly(I:C) agaroses were incubated with or without free poly(I:C) at 0, 150, or 500 μg/ml. Bound proteins were analyzed by immunoblotting with anti-myc. (E) Pull-down competition assays using purified myc-bbeDDX23 or myc-bbeDHX15 in the presence of increasing amounts (0, 1,000, 3,000 μg/ml) of uncoupled low molecular weight (LMW) poly(I:C) or high molecular weight (HMW) poly(I:C). (F) Morphology of primary amphioxus intestinal cells in both resting and upon FITC-labeled poly(I:C) transfection. (G) Expression profiles of bbeDHX9, bbeDHX15, and bbeDDX23 were determined by RT-PCR using poly(I:C) transfected amphioxus intestinal cells. Data are shown as the means ± standard deviations of three samples per treatment. Values were considered to be significant when p < 0.05. The results were confirmed by at least three separate experiments.