Characterization of mass spectrometry samples. (A) Schematic of experimental comparisons. (B) Western blot comparing levels of Flag-AGO2 with endogenous AGO2 in the cytoplasm and with expression in the nucleus. Cytoplasmic and nuclear extracts were separated by SDS-PAGE and blotted with anti-AGO2 antibody. Flag = AGO2 from Flag-tagged AGO2 stable line. Values are fold overexpression as compared to endogenous. (Endog) Endogenous AGO2 from T47D cells. (C) Western blot comparing expression of Flag-tagged AGO2 and endogenous AGO2 in the cytoplasm (left) and in the nucleus (right). Values are distribution levels between the cytoplasm and nucleus. (Cyto) Cytoplasm; (Nuc) nucleus. (D) Fluorescence images of Flag-AGO2. Green is Flag-AGO2, blue is the nucleus (stained with DAPI, 4′,6-diamidino-2-phenylindole). (E) Western blot analysis of cytoplasmic and nuclear fractions from RNase-treated and non-RNase-treated extracts of markers for nuclear, cytoplasmic, and ER proteins. Coomassie staining of Flag immunoprecipitations from cytoplasmic lysate prior to mass spectrometry. The control is using T47D cytoplasmic lysate, the sample is using Flag-AGO2 stable line cytoplasmic lysate. The band at ∼100 kDa is identified as AGO2.
