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. Author manuscript; available in PMC: 2014 Apr 25.
Published in final edited form as: Cell. 2013 Apr 25;153(3):575–589. doi: 10.1016/j.cell.2013.03.024

Figure 1. ADAR1 interacts with Dicer in an RNA binding-independent manner.

Figure 1

(A) Pulldown products of FLAG-tagged RISC member proteins and reciprocal pulldown of FLAG-tagged ADAR1 (F-ADAR1p110) purified from HEK293 cell extracts. Cytoplasmic extract (20 μg) and FLAG-IP peak eluate (15 μl) were examined by immunoblotting analysis. A mock FLAG-IP conducted with untransfected HEK293 cells was used to monitor the background levels of protein that may associate with the FLAG Ab resin (right panels). (B) F-Dicer IP products fractionated by Superose 6 gel filtration column chromatography were analyzed by immunoblotting. The positions of the molecular mass size markers are indicated by open arrowheads. (C) Analysis of recombinant protein interactions in the Sf9 cell co-infection/co-purification system. Various H-ADAR1 expression baculoviruses were co-infected with F-Dicer, untagged TRBP, or F-Ago2 expression viruses. FLAG IP isolated F-Dicer or F-Ago2, and immunoblotting determined their interaction with ADAR1. TALON purifications isolated each H-ADAR1s, and immunoblotting determined their interaction with TRBP. (D) Analysis of recombinant proteins purified from Sf9 cells triple-infected with F-Dicer, H-ADAR1, and HA-Ago2. Immunoblotting determined ADAR1 indirect interaction with Ago2 mediated by Dicer. (E) Analysis of recombinant proteins purified from Sf9 cells triple-infected with F-Dicer, H-ADAR1, and untagged TRBP. FLAG purifications isolated F-Dicer, and immunoblotting determined its relative interaction with ADAR1 and TRBP. (F) Analysis of recombinant proteins purified from Sf9 cells co-infected with F-Dicer and TRBP. FLAG purifications isolated F-Dicer, and immunoblotting determined its relative interaction with TRBP in the absence of ADAR1.