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. 2015 Oct 1;29(19):1998–2003. doi: 10.1101/gad.266486.115

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© 2015 Köhn et al.; Published by Cold Spring Harbor Laboratory Press

This article, published in Genes & Development, is available under a Creative Commons License (Attribution 4.0 International), as described at http://creativecommons.org/licenses/by/4.0/.

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Figure 4. — Y3** associates with the 3′ UTR of histone pre-mRNAs. (A) The copurification of proteins with the indicated Y RNAs, as in Figure 1C, was analyzed by Western blotting with the indicated antibodies. (C) Bead control; (I) input fraction (5% of total). (B) HEK293 cells were transfected with FIP1L1 and/or CPSF4 for 48 h as indicated. The pull-down (PD) of proteins with Y3**, as in Figure 1C, was analyzed by Western blotting. (C) Bead control; (I) input fractions (5% of total). (C) The copurification of the indicated mRNAs with biotinylated Y3 or Y3** was analyzed by RT-qPCR. The ratios of mRNA levels (pull-down/input) were determined by the ΔC_T method. Streptavidin resin served as a negative control (C). Error bars indicate the SD of at least three independent analyses. (D, top panel) Biotinylated H3A-derived transcripts used as bait for the copurification of Atto680-labeled Y3** in pull-down studies are depicted relative to the full-length H3A histone pre-mRNA using color coding. Copurification of Atto680-labeled Y3** and pull-down of biotinylated histone transcripts were monitored by infrared scanning (Y3**) or Syto60 staining (histone RNAs) of TBE-urea gels, respectively. Streptavidin resin (C) served as negative control. (I) Input (2% of total). (E) Subcellular localization of the indicated ncRNAs was analyzed by cell fractionation and Northern blotting of total (T), nuclear (N), or cytoplasmic (C) RNA isolated from human HEK293 or murine MC57G cells. U11 and 7SL served as controls for the enrichment of nuclear or cytoplasmic RNAs, respectively. (F) Chromatin immunoprecipitation (ChIP) analyses were performed in HEK293 stably expressing GFP-tagged human Mini-Flash (GFP-hMF). Beads only (C) served as a negative control, and Histone H3 served as a positive control. (Top panel) Eluted DNAs were PCR-amplified (HIST2H3A, HIST1H2AC, and intergenic DNA) and analyzed together with the input (I) on agarose gels. (Bottom panel) Coprecipitated RNAs were subjected to 3′-RACE, PCR-amplified, and analyzed by Southern blotting using the indicated probes.