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. 2008 Mar 26;3(3):e1864. doi: 10.1371/journal.pone.0001864

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This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.

This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration, which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.

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(A) Cells were left untreated (Untr.) or treated with puromycin (Puro., 200 µM, 1 hr); cytoplasmic lysates were prepared and fractionated through sucrose gradients. Top, representative miR-24 levels in each fraction, visualized after RT-PCR (30 cycles) and electrophoresis (1.5% agarose). Bottom, miR-24 levels in each fraction, quantified by RT-qPCR analysis and represented as % of the total miR-24 (semilogarithmic scale). (B) Representative gradient profiles. (C) By 48 hr after transfection of pre-miR-24 or control (Ctrl.) siRNA (100 nM), miR-24 and U6 snRNA levels were visualized on agarose gels (left), quantified by RT-qPCR, and normalized to 5S (right). Data (means+SD) represent the fold differences in miR-24 levels between the transfection groups. (D) HeLa cells were transfected with a plasmid expressing either pTRESNeo (V) or pIRESNeo-HA-Ago1 (HA-Ago1); the specific IP of HA-Ago1 was assessed by Western blot analysis of the IP reaction products. Forty-eight hr after transfection of HeLa cells with plasmid (V or HA-Ago1) and either control (siRNA) or pre-miR-24 RNA, RT-qPCR analysis was used to test the association of p16 mRNA with the RISC complex in the V and HA-Ago1 groups after HA IP. Graphs show the means+SD from 3 independent experiments.