Fig 1: RNase H1 Protein Is Present in the Cytosol and the Reduction of RNase H1 Dramatically Decreases ASO Activity(A) Cytosolic, mitochondrial, and nuclear fractions were prepared, and proteins of each fraction from an equal number of cells were analyzed by SDS-PAGE and western blot assay. The membrane was probed for the following, using specific antibodies: RNase H1; nuclear proteins PSF, P54nrb, and Lamin A; mitochondrial proteins P32, GRSF1, and Hsp60; and cytoplasmic protein GAPDH. The RNase H1 protein level was quantified using ImageJ software (NIH). The mean levels and SDs from three independent experiments are shown below the lanes. (B) qRT-PCR (left) and western blot (right) analyses for the levels of RNase H1 and RNase H2 mRNAs and proteins in HeLa cells treated with specific siRNAs or a control luciferase siRNA for 60 hr. GAPDH was probed in western blot analysis and served as a loading control. (C–H) Control, RNase H1-, and RNase H2-siRNA-treated cells, as used in (A), were treated for 4 hr with different ASOs, and the levels of the targeted MALAT1 (C), SOD1 pre-mRNA (D), Ago2 (E and F), NCL1 (G), and Drosha (H) RNAs were determined by qRT-PCR. Error bars are SDs from three experiments. p values were calculated using the F test (curve comparison between control and H1-reduced cells).