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. Author manuscript; available in PMC: 2019 Feb 16.
Published in final edited form as: J Mol Biol. 2017 Jul 10;430(4):537–553. doi: 10.1016/j.jmb.2017.06.023

Figure 6.

Figure 6

Co-IP between Rev and DEAD proteins. (a) Schematic diagrams of the plasmids used for cell line engineering and lentivirus production. Top: plasmid for Flp recombinase-mediated integration and its expression is under the control of a CMV/Tetracycline-regulated promoter. Flp-In T-Rex HeLa cells were transfected with this plasmid and selected with Hygromycin to establish stable T-Rex-GagRRE HeLa cells. The bottom three diagrams represent the lentivectors containing indicated proteins. (b) Western blot analysis of co-IP between Rev and DEAD proteins. To examine the interactions of DEAD helicases and Rev, T-Rex-GagRRE HeLa cells were transduced with lentiviruses of Rev-GFP-V5 for DDX3 IP, or cotransduced with lentiviruses of mCherry-DDX1-V5, or mCherry-DDX21-V5 for 24 hr followed by Doxycycline addition for another 24 hr for DDX1 or DDX21 IP. Cell lysates with and without RNase treatment were incubated with indicated antibodies, and then analyzed by western blotting with indicated. Cells expressing Rev(V16D)-GFP-V5 were analyzed with Rev-GFP-V5 in parallel. Asterisk indicates overexposed samples for better visualization.