FIG 4.

dsVEGF706 is preferentially bound to the sense NcVEGF RNA. PC-3 cells were transfected either with control dsRNA biotinylated on the 3′ ends of both sense and antisense dsCT(S+AS)biot or a dsVEGF706 biotinylated on the 3′ ends of either the sense strand [dsVE(S)biot] or the antisense strand [dsVE(AS)biot]. After 72 h, the cells were fixed and treated as described in RIP experiments using streptavidin-agarose beads to pull down NcVEGF or GAPDH transcripts. Both total RNAs (inputs) and streptavidin pulled-down RNAs were used in standard reverse transcription reactions, followed by semiquantitative (lower panel) and quantitative (top panel) PCRs to amplify either NcVEGF (S788/R588 or S788/R706) or GAPDH. Also, 10% of the cell lysates was kept to obtain the RNA used to generate the input lanes, and nonspecific pulldown of GAPDH mRNA was used to normalize these assays. The histogram shows the NcVEGF/GAPDH qPCR ratio after biotin pulldown (left side) and in the input lanes (right side). To quantify the specific pulldown of NcVEGF using biotin-labeled dsVEGF706, the ratio obtained from dsCT(S+AS)biot-transfected cells was arbitrarily set to 1. The values are means ± the SD of three independent experiments with each sample run in duplicate. *, P ≤ 0.05; **, P ≤ 0.005. The lower panel shows the results from a representative semiquantitative PCR after biotin pulldown with either qPCR-VEGF (R588/S788) or GAPDH primers.