Ago2 is important for snoMEN RNA interference.

<p>(<b>a</b>) HeLa cells were transfected with siRNA oligonucleotides for Ago1, Ago2, and Upf1. Whole cell lysates were prepared 48 hours after transfections and analysed by western blot using the indicated antibodies. (<b>b</b>) HeLa cells were transfected with siRNA oligonucleotides prior to pri-miR21-snoMEN transfection for 24h. Total RNA from HeLa cells was harvested and qPCR was performed, followed by cDNA synthesis, using matured miR21 specific primer and universal primer provided by the PerfeCta SYBR Green qPCR kit (Quanta Biosciences, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138668#sec008" target="_blank">methods</a>). (<b>c</b>) HeLa cells transfected with pri-miR21-snoMEN for 24 hours prior to protein extraction. The same amount of proteins/RNAs from each subcellular fraction was loaded on each lane and analysed by western blot/Northern blot using the indicated antibodies (Ago1, Ago2, Upf1) / RI-labelled probes (snoMEN, tRNA). The fractionation markers, i.e. Tubulin (Cytoplasm), Lamin A/C (Nucleoplasm), Fibrillarin (Nucleoli), were tested to evaluate fractionation quality. (<b>d</b>) Images show localisation pattern of DNA (DAPI, Blue), a transfection FP-marker of pri-miR21-snoMEN (mCherry, Red) and the indicated proteins, i.e. Ago1, Ago2 and Upf1 (FITC, Green). Scale bar is 10 μm. Arrows and arrowheads show transfected and non-transfected cells, respectively. Specificity of each antibody was also verified by siRNA transfections (<b>Fig K</b> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138668#pone.0138668.s001" target="_blank">S1 File</a>). (<b>e</b>) HeLa cells were transfected with pri-miR21-snoMEN for 24 hours prior to fractionation. Cell lysates of each subcellular fraction were prepared and immunoprecipitated with either normal mouse IgG, or anti-Ago2 antibodies. Total protein was isolated from precipitates and resolved by SDS-PAGE and then analysed by western blotting using an Ago2 antibody to validate the efficiency of purification. In parallel, total RNA was also isolated from each precipitate and qRT-PCR was performed using the specific primers indicated.