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. 2017 Jul 29;45(18):10436–10451. doi: 10.1093/nar/gkx667

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© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 5. — ADARs interact with Dicer to promote miR-27a expression independent of RNA editing/dsRNA binding activities. (A–C) The qRT-PCR measurement of mature miR-27a (A), pri-miR-27a (B), and pre-miR-27a (C) in Huh-7 cells upon overexpression of empty vector (EV) or different ADARs expression constructs including the wild-type, DeAD and EAA mutants. Data are presented as the mean ± s.e.m. of triplicates from a single experiment and representative of 3 independent experiments (*P < 0.05; **P < 0.01; ***P < 0.001). Statistical significance is determined by unpaired, two-tailed Student's t test. (D) Reciprocal immunoprecipitation (IP) of ADARs and Dicer is performed in HEK293T cells overexpressing FLAG only or FLAG-tagged wild-type and mutant form of ADARs, using anti-FLAG antibody conjugated magnetic beads (IP: FLAG; top panel), or Dicer-specific antibody (IP: Dicer; bottom panel) or mouse IgG (IP: IgG; bottom panel). Western blot analyses of the indicated proteins in FLAG, Dicer or mouse IgG-IPed products are conducted. (E) The purity of cytoplasmic (Cyto) and nuclear (Nu) fractions of HEK293T is verified by western blot analyses of ADAR1, ADAR2, Dicer, Lamin A/C, Fibrillarin and α-tubulin. (F–H) Reciprocal IP of endogenous ADARs and Dicer is conducted in the cytoplasmic (Cyto) and nuclear (Nu) fractions of HEK293T cells using an Dicer (F), ADAR1 (G), ADAR2 (H)-specific antibody (IP: ADAR1, ADAR2 or Dicer) or mouse IgG (IP: IgG). Western blot analyses of the indicated proteins in ADAR1, ADAR2, Dicer or mouse IgG-IPed products are conducted. Input indicates 5% of each fraction or total lysates from HEK293T cells. Total lysates (Total) are included in the experiments describe in (E–H) as positive controls.