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. Author manuscript; available in PMC: 2012 Feb 18.
Published in final edited form as: Mol Cell. 2011 Feb 18;41(4):409–418. doi: 10.1016/j.molcel.2011.01.022

Figure 4. siRNA knockdown of PAF1c inhibits 3′end processing of reporter mRNA.

Figure 4

(A) Schematic of reporter plasmid pGL3G5E4 containing GAL4 binding sites upstream of E4 core promoter, Luciferase coding sequences and SVL poly(A) site downstream.

(B) RNase protection assay of transcripts isolated from 293T cells treated with siRNA targeting PAF1c subunits. The reporter plasmid was transfected into 293T cells pre-treated with siRNAs targeting PAF1c subunit(s) as indicated. The next day, cells were harvested and total RNAs were obtained using Trizol (Invitrogen), fractionated into nuclear and cytoplasmic fractions and 3′ cleavage efficiency was examined by RNase protection assays. Quantitation was performed with ImageJ. Ethidium bromide staining of 5S rRNA (lower panel) indicates uniform recovery between samples.