2004;24:2637C2648. for Rev-mediated appearance of the HIV-1 genome. DH5, were transformed with pGEX-5X1-Rev, pGEX1T-Pur [Gallia et al., 1998]. GST pull-down assay was performed according to the procedures described previously [Kaniowska et al., 2006]. RNA Preparation and Northern Blot Analysis Orientin Total RNA was isolated using RNeasy kit (Qiagen, Valencia, CA, #74104) according to the manufacturers directions. Preparation of cytoplasmic RNA was based on the protocol provided with RNeasy kit and using RLN buffer containing 50 mM Tris-HCl, pH 8.0, 140 mM NaCl, 1.5 mM MgCl2, 0.5% (v/v) Nonidet P-40 (1.06 g/ml), 1,000 U/ml RNase inhibitor, 1 mM DTT. Ten micrograms of RNA were fractionated on 1.2% agarose, 0.4% formaldehyde, 1 morpholinepropanesulfonic acid (Mops) gel and transferred to a Hybond-N nylon membrane (Amersham, Piscataway, Orientin NJ). For the detection of CAT RNAs, the membranes were hybridized with [32P]-labeled CAT DNA probe obtained by digestion of pIIIAR plasmid with em BgI /em II and em Nco /em I (850 bp). For the detection of RRE containing RNAs, the filters were probed with a PCR-amplified and [32P]-labeled DNA fragment consisting of nucleotides 1-234 of the RRE. Radiolabeled DNA probes were prepared with Random Primed DNA Labeling Kit (Roche Molecular Biochemicals, AF-9 Indianapolis, IN), followed by removal of unincorporated radionucleotides with MicroSpin?G-50 columns (Amersham). Relative levels of mRNA were determined by using the housekeeping gene GAPDH as an internal standard. RNA Immunoprecipitation and RT-PCR Nuclear and cytoplasmic protein extracts from cells transfected with pcDNA3.1-SD4*-luciferase-RRE-SA7 vector along with Pur and/or Rev expressing plasmids were prepared and precleared with protein A sepharose beads by 1 h incubation at 4C. The resultant lysates were incubated with protein A sepharose beads and anti-T7 antibody against Pur. In control samples, normal mouse serum was added instead of anti-T7 antibody. For RT control Rev expressing extracts were immunoprecipitated with anti-myc antibody. After overnight incubation at 4C, the beads were washed four times (3 min each) with HNTG buffer (20 mM HEPES, pH 7.5/150 mM NaCl/0.1% Triton X-100/10% Glycerol, protease inhibitor cocktail (Sigma, P8340) and 0.2 mM Na-orthovanadate) and resuspended in 150 l of buffer 2 (50 mM Tris-HCl, pH 7.0/5 mM EDTA/1% SDS/10 mM DTT). Then RNA was extracted using phenol TE buffer pH 4.5 saturated/chloroform/isoamyl (25/24/1) and chloroform/isoamyl (24/1). Extracted RNA was precipitated by incubation with 7.5 M ammonium acetate/ethanol (0.5/2.5, V/V) and GlycoBlue (Ambion, Austin, TX) for 30 min at -70C followed with washing in 70% ethanol, drying and rehydration in 20 l of nuclease free water. Samples were treated with DNAse1 in the presence of RNase inhibitor (Roche) for 1 h at 37C followed with phenol/chloroform extraction and precipitation as described above. RT reaction was performed using random primers (p(dN)6; Roche) and M-MuLV RT enzyme. For PCR, RRE specific primers and FailSafe? PCR reagents (Epicentre, Madison, WI) were used. DNA products generated as a result of RT-PCR were analyzed by 2% agarose gel electrophoresis. Fluorescent Technique U-87MG cells (1 105) were transfected with 5 g of GFP-Pur or Rev-Red plasmids, alone or in combination, then seeded in poly-l-lysinecoated glass chamber slides, and after 16 h incubation, cells were fixed in 4% paraformaldehyde in 1 PBS. Cells were then washed in PBS, and proteins were visualized for green or red fluorescence. Fluorescent images were captured using an inverted fluorescent Nikon microscope with deconvolution software (Slide-Book 4.0.1.34; Intelligent Imaging, Denver, CO). RNA Electrophoretic Mobility Shift Assay Orientin The RRE-containing RNA probe was in vitro transcribed using pBluescript IIKS(+) RRE 1-234 and pBluescript IIKS(+) RRE 34-110 plasmids as DNA Orientin template. In vitro transcription reaction was performed by incubating the above described template DNAs, T3 RNA polymerase, transcription optimized 5 buffer, DTT 100 mM, recombinant RNasin ribonuclease inhibitor, rATP, rGTP, rCTP (2.5 mM each) and [-32P]-UTP for 1 h at 37-40C. For RNA-protein interaction studies 0.2 M of total recombinant proteins GST, GST-Rev, GST-Pur were incubated with 100,000 cpm of [-32P]-UTP RNA probe for 15 min at room temperature in 20 l binding buffer containing 12 mM HEPES (pH 7.9), 4 mM Tris-HCl (pH 7.5), 60 mM KCl, 5 mM MgCl2, 0.8 mM dithiothreitol (DTT), 0.5 g of poly[dI-dC] as a nonspecific competitor, 10% glycerol and RNase inhibitor. Reaction products were analyzed on a 1% agarose gel followed by autoradiography. Antibodies Anti–tubulin, clone B512 was obtained from Sigma-Aldrich. Anti-myc antibody was purchased from Invitrogen and anti-T7 tag antibody was from Novagen.