[PubMed] [Google Scholar] 10

[PubMed] [Google Scholar] 10. the glutathione-sepharose beads precleared with control bacterial extract to remove nonspecific proteins. Following incubation of the extract with the beads for 12 h at 4C, the beads were washed extensively and the bound proteins were released from the beads by boiling in the presence of SDS buffer. The released proteins were subjected to SDS-PAGE analysis and Coomassie staining to check the purity of the glutathione bead-bound GST, GST-N and GST-P proteins. In each case, the amounts of GST and GST fusion protein(s) bound to the glutathione beads were similar and the proteins were homogeneous. To study in vitro binding of purified -catenin with GST-N and GST-P proteins, Desonide equal amount of Desonide purified his-tagged -catenin was added to the washed GST, GST-N and GST-P protein-bound glutathione beads. Following incubation at 4C for 12 h, the beads were washed extensively and the bound proteins were released in the presence of SDS sample buffer. The released proteins were subjected to 7.5% SDS-PAGE and Western blot analysis with -catenin antibody. A portion of the glutathione-bound proteins was also subjected to Western blot analysis with GST and HPIV-3 RNP antibodies to monitor the expression of GST, GST-N, and GST-P during the in vitro binding studies. Double Labeled Immunofluorescence Confocal Microscopy A549 Desonide cells grown on coverslips were infected with HPIV-3 at 1 pfu/cell. At 36 h postinfection, the cells were washed with phosphate-buffered saline followed by fixation with 3.6% paraformaldehyde and permeabilization with 1% Triton X-100. The fixed cells were treated with a mixture of rabbit anti-RNP and mouse monoclonal anti–catenin antibodies followed by incubation with Texas Red conjugated anti-rabbit and FITC conjugated Opn5 anti-mouse secondary antibodies. The coverslips were finally washed, mounted, and examined using a Leica CLSM confocal laser-scanning microscope as described previously (7,21). In Vitro Transcription Fifty microliters of in vitro transcription reactions (17) was prepared in the presence of 100 mM HEPES-KOH (pH 8.0), 100 mM KCl, 5 mM MgCl2, 1 mM DTT, 1 mM each of ATP, GTP, and UTP, 10 M CTP, 15 Ci of [-32P]CTP, 25 units of RNase inhibitor, and 2 g of purified HPIV-3 RNP. The reaction was carried out in the presence or absence of immunoprecipitated -catenin and/or actin pellet (P) (actin or -catenin from uninfected A549 cell lysate bound to washed and precleared protein A-sepharose beads) and -catenin immunodepleted supernatant (S) (uninfected A549 cell lysate not bound to the -catenin immunopellet), along with control rabbit serum immunoprecipitated pellet and immunosupernatant. In another set of transcription reaction, actin and -catenin immunopellets were added together to the reaction mixture. For these experiments, uninfected A549 cell lysates (100 g protein/reaction) were immunoprecipitated with the antibodies in the presence of washed protein A-sepharose beads. The immunopellets comprising the proteins bound to the beads were extensively washed prior to using them for in vitro transcription assay. The immunodepleted -catenin supernatant obtained following incubation of the cell lysate with -catenin antibody bound beads was further immunoprecipitated with -catenin antibody to remove residual -catenin protein from the supernatant. The doubly immunoprecipitated supernatant was used for the in vitro transcription assay. In addition, the in vitro transcription assay reaction was carried Desonide out in the absence or presence of two different concentrations (1 and 0.5 g) of purified recombinant his-tagged -catenin. As a control, reaction was also performed in the presence of purified RNP alone. The reaction mixture was incubated at 30C for 3 h, and the in vitro synthesized 32P-labeled RNA products were purified by phenol extraction and ethanol precipitation and analyzed by 5% polyacrylamide-urea electrophoresis, followed by autoradiography. RESULTS Association of em /em -Catenin With HPIV-3 RNP Earlier studies have demonstrated that optimal transcription of HPIV-3 RNP, associated with the actin microfilament, required not only actin but additional actin-bound host protein(s) (15). Based on this observation, it was speculated (21) that actin-interacting cellular transcriptional activators may be required for actin to fully function as the transcriptional activator of HPIV-3 genome. -Catenin, an actin-bound protein, became our logical choice as one of the actin-bound transcriptional activators because it constitutes one of the abundant proteins that are bound to actin filament in the cytoplasm and it posses transactivation function (4,13). Thus, to initially investigate whether -catenin indeed interacts with the viral RNP, HPIV-3 RNP isolated from infected human lung epithelial A549 cells and purified HPIV-3 virions were subjected to Western blot analysis with -catenin antibody. We utilized human lung epithelial A549.