Figure 1

HIV-1 TAR miRNAs are loaded into Argonaute complexes. Stable HEK 293 cells expressing Flag-Argonaute 1–4 proteins were transfected with either U6-shNEG or U6-TAR or not (mock) for 48 hours. Protein samples were collected (input) and immunoprecipitated (IP) by an anti-Flag antibody. After the last wash, ¼ of the IP sample was kept for protein analysis and ¾ for RNA analysis. All solutions were prepared with DNA/RNA nuclease free water and contain protease and RNAse inhibitors. A) Western blot analysis of Argonaute (Ago) proteins from transfected samples. Input and IP samples were separated on a 10% polyacrylamide gel and immunoblotted with anti-Flag antibody. B) Northern blot of miRNAs expressed from U6-shNEG or U6-TAR transfected cells using probes against NEG-3p or miR-TAR-3p miRNAs. The probe against U6 RNA was used as a loading control for input and IP samples.