TNPO3 Interaction with retroviral integrases. (a) Human 293T cells, which endogenously express TNPO3, were transfected with different amounts of the indicated mammalian codon-optimized FLAG-tagged retroviral integrases (IN). Twenty-four hours following transfection cells were lysed in extraction buffer (400 mM NaCl, 0.5% Triton X-100, 50 mM Tris-HCl, , 2 mM MgCl2, 5% glycerol and protease inhibitors (Roche)). Subsequently, extracts were treated with DNAase and precleared using protein-A agarose beads (Sigma) at 4°C for 1h. Small aliquot of the initial extract was analyzed by Western blot (WB) using anti-TNPO3 antibodies (INPUT). Subsequently, the extracts were used to immunoprecipitate (IP) the different retroviral integrases using anti-FLAG antibodies. FLAG-peptide eluted complexes were analyzed by WB for the presence of TNPO3 and using anti-TNPO3 and anti-FLAG antibodies, respectively. (b) As a positive control we assayed the known ability of HIV-1 integrase to interact with LEDGF/p75. For this purpose, HA-tagged LEDGF/p75 (LEDGF-HA) was cotransfected together with FLAG-tagged HIV-1 integrase and immunoprecipitated using anti-FLAG beads. Eluted complexes were analyzed for the presence of LEDGF/p75 and HIV-1 integrase by WB using anti-HA and anti-FLAG antibodies, respectively. Similar results were obtained in three independent experiments, and the results of a representative experiment are shown.
