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Analysis of pre-rRNA transcription and processing in mouse Dicer+/− and Dicer−/− ES cells.

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posted on 2010-08-13, 01:49 authored by Lasse Sinkkonen, Tabea Hugenschmidt, Witold Filipowicz, Petr Svoboda

(A) Incorporation of 3H-methyl-label into total cell RNA. Mouse Dicer+/− and Dicer−/− ES cells were cultured for 30 min in the presence of [3H-methyl]-methionine. Incorporation measured for Dicer+/− ES cells was set as 100%. The values represent means (+/− SD) from samples collected at 0, 30 and 60 min of after labeling with [3H-methyl]-methionine. (B) Analysis of pre-rRNA processing by agarose gel electrophoresis. Equal amounts of RNA isolated from cells cultured for 30 min in the presence of [3H-methyl]-methionine and chased with unlabeled methionine for 0, 30 and 60 min were separated on agarose gel and RNA was visualized by fluorography. (C) UBF association with rRNA promoter is not affected in Dicer−/− ES cells. The ChIP was performed with UBF antibody [49] as described in Figure 2E. Results are shown relative to the 1∶100 dilution of respective input DNAs and represent means (+/− SEM) of at least 3 independent experiments.


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