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. 2015 Feb 26;43(5):2638–2654. doi: 10.1093/nar/gkv138

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© The Author(s) 2015. 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/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 1. — Identification of Ago2 and DDX6 as novel TRIM32-associated proteins via mass spectrometry. (a) Scheme depicting the experimental design of the mass spectrometry approach. TRIM32 was precipitated from NSCs either grown under maintenance conditions or 3 days after the induction of neuronal differentiation using an anti-TRIM32 antibody. Proteins that coprecipitated with TRIM32 were analyzed by mass spectrometry, which identified Ago2 and DDX6 as potential TRIM32 interaction partners (the full list of identified proteins is provided in Supplementary Table S1). (b) HEK293T cells were transfected with the indicated constructs and the different FLAG-tagged Ago proteins were immunoprecipitated with an anti-FLAG antibody. Ago-associated TRIM32 was detected with an anti-TRIM32 antibody. TRIM32 and FLAG-tagged Ago proteins were detected with specific antibodies as indicated for the immunoprecipitation (IP) and the lysate. Note that although Ago4 was detectable after enrichment via precipitation, no Ago4-expression could be detected within the lysate (asterisk). (c) HEK293T cells were transfected with the indicated constructs and Venus-FLAG-tagged DDX6 was immunoprecipitated with an anti-FLAG antibody. DDX6-associated TRIM32 was detected with an anti-TRIM32 antibody. TRIM32 and Venus-FLAG-tagged DDX6 were detected with specific antibodies as indicated for the IP and the lysate. (d) HEK293T cells were transfected with the indicated constructs and FLAG-tagged Ago2 was immunoprecipitated with an anti-FLAG antibody. Ago2-associated DDX6 was detected with an anti-DDX6 antibody. DDX6 and FLAG-tagged Ago2 were detected with specific antibodies as indicated for the IP and the lysate. In (b–d), GAPDH western blots were used as loading control.