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. 2015 Jul 15;5(3):1441–1466. doi: 10.3390/biom5031441

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© 2015 by the authors; licensee MDPI, Basel, Switzerland.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).

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Validation of double-tagged DDX6 construct to ensure highly specific interaction screen. (A) Exogenously expressed DDX6 has physiological expression levels. Western blot analysis of whole cell lysates from HEK-293 cells (lane 1) or HEK-293 cells containing the DDX6-FLAG-HA construct under control of a tetracycline-inducible promoter (lanes 2 and 3). DDX6-FH = FLAG-HA-tagged DDX6 construct inducibly expressed from 293-DDX6-FH cells. End-DDX6 = endogenous DDX6; (B) Exogenously expressed DDX6 localizes to the expected cellular compartments. Immunofluorescence analysis of HEK-293-DDX6-FLAG-HA cells, in the absence or presence of doxycycline, with antibodies against endogenous DDX6 or the HA epitope tag; (C) Quantification of the average number of P bodies per cell in HEK-293 cells as compared to 293-DDX6-FH cells in the absence or presence of doxycycline.