Protein concentration in purified IN preparations was determined by micro Bradford assay. Fragments were aliquoted natural compound library and frozen at 280uC. Integrase Activity Assays DNA duplexes for examining integrase activity. Integrase activities were examined using synthetic DNA duplexes. DNA duplex U5 consisting of the oligonucleotides U5B and U5A, which mimicked the end of HIV 1 U5 LTR, served as a substrate for 39 processing activity. Duplex U5 2, formed by U5A and U5B 2, was used as a substrate for strand exchange and duplex Ran formed by oligonucleotides RanB and RanA, to confirm the specificity of 39 processing. To evaluate integrase catalytic activities, the oligonucleotides U5B, U5B 2, and RanB were labeled using 50 mCi of ATP and T4 polynucleotide kinase. After 1 hour of incubation at 37uC, EDTA was put into the final concentration of 50 mM, and the reaction mixture was heated for five minutes at 65uC to inactivate the kinase. Described resonance oligonucleotides were formulated with equimolar amounts of unlabeled contrasting oligonucleotides and annealed by first heating for three minutes at 90uC and then cooling slowly to room temperature. Resulting duplexes were purified using Micro Bio Spin tips G 6. 39 end control and strand transfer reactions. All assays were performed as described previously. DNA duplexes were incubated for 2 hours with 100 nM IN in 20 ml of the buffer containing 20 mM Hepes, pH 7. 2, 7. 5 mM MgCl2, and 1 mM DTT, at 37uC. DNA fragments were precipitated with ethanol and separated in denaturing two decades polyacrylamide ties in. Ties in were quantified with Image QuantTM 4 and analyzed on the Storm 840TM PhosphorImager. 1 application. Integrase task was defined as percent substrate converted to a solution, activities of IN variants were quantified relative to IN a values. Each test was repeated at least three times with convergent results. Eukaryotic Expression of Integrases HEK293, supplier Oprozomib NIH3T3 and HeLa cells were cultured in the Dulbecco s altered Eagle s medium supplemented with ten percent fetal bovine serum at 37uC in five full minutes CO2 humidified atmosphere. Cells were transfected with pVaxIN a, pVaxIN in, pVaxIN in e3, or empty vector pVax1 using Lipofectamine LTX. At time 48 post transfection, cells were harvested, lysed and analyzed by electrophoresis in 127-inch SDS PAAG with subsequent Western blotting, applying for staining polyclonal anti IN rabbit sera. Binding was visualized by secondary HRP conjugated anti rabbit antibody. The membrane was created utilizing the ECL plus american blotting detection system. To normalize for the sum total protein content, membranes were stripped based on the ECL process and re-stained with monoclonal mouse anti actin antibody, accompanied by the HRPconjugated anti mouse antibody.