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. 2010 Jun 15;18(9):1675–1682. doi: 10.1038/mt.2010.123

Figure 2.

Figure 2

U1-antisense expression in HeLa cells. (a) Schematic representation of pRRL/U1-antisense constructs. The U1-antisense expression cassettes were cloned in the dU3 region of the downstream LTR of the pRRLSIN.cPPT.PGK/GFP.WPRE lentiviral backbone. (b) HeLa cells were transfected with the different antisense constructs together with the U16-RBE plasmid (expressing a 143-nt long modified U16 snoRNA). Northern blot analysis was performed with probes against: U1 snRNA (panel U1), U16-RBE (panel RBE), and U2snRNA (panel U2). The two latter hybridizations are used to normalize for transfection efficiency and as loading control, respectively. The filter was cut so as to exclude the endogenous U1 snRNA hybridization. On the side, the molecular sizes are reported. (c) Nuclear extracts were prepared from the 5′3′esx sample from the experiment in a and immunoprecipitated with anti U1-70K antibodies. The RNA, extracted from the Ippt pellet, was analyzed for the presence of the 5′3′esx transcript by reverse transcriptase-PCR with primers shown on the right panel. GFP, green fluorescent protein complementary DNA; hPGK, human phosphoglycerate kinase promoter; nt, nucleotide.