Xenbase Image ID: 127866

Figure 7. AGO2 interacts substantially with FXR1 and associates with activated but not repressed targets in G0 human cell nuclei.All interactions were observed after in vivo formaldehyde crosslinking4054 followed by nuclear-cytoplasmic fractionation and immunoprecipitation. (a) Immunoprecipitation of AGO2 (Wako) or control IgG from nuclear and cytoplasmic extracts of proliferating (Cycling) and G0 THP1 cells followed by RT-PCR analysis demonstrated that TNFα mRNA was specifically associated with AGO2 exclusively in G0 nuclei after 4 hrs of serum-starvation induction of G0 (TNFα, first panel, 4 hrs G0) and also in the G0 cytoplasm later at 8 hrs of serum-starvation (TNFα, third panel, 8 hrs G0). Cyclin E mRNA, which is repressed in G034, served as a control and is not present in the nuclear complex but in the G0 cytoplasmic complex (Cyclin E, second panel, 4 hrs G0; the mRNA is downregulated and below detection by 8 hrs). Input (INP) = 10% of the sample. (b) G0 HEK293 cells were transfected with reporters that bear the AU-rich target sequence of TNFα mRNA (TNFα ARE, sufficient for activation in G05455) or a mutated sequence (mtARE, non-functional for activation)5455. Immunoprecipitation of AGO2 or control IgG from nuclear and cytoplasmic extracts from transfected late G0 cells followed by RT-PCR analysis demonstrated that the ARE reporter was associated substantially with AGO2 in G0 nuclei, with some association in G0 cytoplasm as expected at late G0, similar to (a). No association was observed with the mutated (mtARE) reporter; tRNAlys and Renilla served as controls. The average values of three replicates with standard deviations as error bars are shown. (c) Western analyses of immunoprecipitates from 4hr G0 and cycling (Cyc) samples from (a) using anti-AGO2 (Millipore) and anti-FXR1. Input (INP) = 10% of the sample. A non-specific band immunoprecipitated with anti-AGO2 and IgG in the AGO2 Western blot analysis below the specific AGO2 band. Anti-FXR1 recognizes several bands in the Input lane representing seven isoforms and modifications. In G0 cytoplasmic fractions (Cyto-G0), the antibody immunoprecipitated less AGO, possibly due to the epitope being masked. In cycling cell cytoplasmic fractions (Cyto-Cyc), AGO is immunoprecipitated at equal levels to that in the nucleus but FXR1association is clearly reduced. AGO2-FXR1 complex is substantially present in the nucleus. Image published in: Truesdell SS et al. (2012) Copyright © 2012, Macmillan Publishers Limited. Creative Commons Attribution-NonCommercial-NoDerivatives license Larger Image Printer Friendly View

Return to previous page