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Efficiency of RNAi-mediated knockdown of <i>CSN3</i> and <i>CSN5</i>.

By Eunju Kim (342950), Se-Jin Yoon (342951), Eun-Young Kim (16515), Yunna Kim (342952), Hyun-Seo Lee (342954), Kyeoung-Hwa Kim (342956) and Kyung-Ah Lee (251243)

Abstract

<p>(A) Determination of the critical time point for complete knockdown of <i>CSN3</i> and <i>CSN5</i> after RNAi. Oocytes were collected every 2 h after RNAi, and <i>CSN3</i> and <i>CSN5</i> mRNA was assessed by RT-PCR. (B) Specific suppression of <i>CSN3</i> or <i>CSN5</i> mRNA by RNAi. The other CSN subunits were not affected by RNAi treatment. <i>H1foo</i> was used as an internal control of oocytes, and exogenous <i>GFP</i> mRNA was used as an external control. (C) Western blot analysis of CSN3 and CSN5. The blot was incubated with each antibody using 100 oocytes. Numbers on the left side of the band indicate the sizes (kDa) of the protein markers, while arrows indicate the specific proteins. β-Actin was used as a loading control. (D) Protein knockdown after RNAi. Levels of CSN3 and CSN5 protein in RNAi-treated oocytes were determined using Western blot analysis. Proteins were extracted from 100 MI oocytes for each lane. (E, F) Bar graphs show the relative mRNA and protein levels after RNAi. The mRNA level was calculated with quantitative real-time RT-PCR using single-equivalent oocyte cDNA, while protein level was calculated by measuring the density and area of the bands. The mRNA and protein levels are presented relative to those of control oocytes. Asterisks indicate statistically significant differences compared to that of control or buffer group (p<0.05).</p

Topics: Biochemistry, Cell Biology, Molecular Biology, Developmental Biology, rnai-mediated, knockdown
Year: 2013
DOI identifier: 10.1371/journal.pone.0025870.g002
OAI identifier: oai:figshare.com:article/399414
Provided by: FigShare
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