Cells were harvested in 1% NP-40 lysis buffer with protease inhibitors and the lysates were cleared by centrifugation for 8 min at 14,000 rpm. half-life. The differential effect of these IFN-Cinducible proteins on MHC class I processing may have a decisive influence on the quality of the CTL immune response. 0111:B4, Difco Laboratories). Ficolled cells were washed and used as targets in CTL assays. The env-specific CTL clone 10B6, generated against Moloney MuLV in a B6.CH-2bm13 mouse, recognizes the SSWDFITV epitope presented by H-2Kb (env; amino acids 189C196) as previously described 23. H-2DbCrestricted CTL clone 1 specific for the gag-leader peptide (CCLCLTVFL; amino acids 75C83) was generated against Moloney MuLV in a B6 mouse, as previously described 24. All cell lines were cultured in Iscove’s modified Dulbecco’s medium FJH1 (Biowhittaker Europe), supplemented with 8% heat-inactivated FCS (GIBCO BRL), 2 mM L-glutamine (ICN Biomedicals), 100 IU/ml penicillin (Yamanouchi Pharma), and 30 M 2-ME (Merck) at 37C in humidified air with 5% CO2. B6 mice were bred under specific pathogenCfree conditions in the TNO-PG breeding facility. TAP?/? mice were purchased from The Jackson Laboratory (B6/129 TAP?/?). DNA Constructs and Generation of Transfectant Cell Lines. The eukaryotic expression plasmids pTET-splice and pTET-tTAk, containing the tetracycline-regulated transcription activator tTAk, have been described elsewhere 25. Generation of the MEC217 cells expressing inducible levels of the cDNAs of murine (H-2b haplotype) LMP2, LMP7, and MECL-1 was described recently 26. cDNAs of the murine PA28 and (H-2b haplotype) were cloned into the EcoRI/EcoRI and SalI/EcoRV sites of the pTET splice vector using standard procedures. The PA28 transfected cells were established by calcium-phosphate precipitation. In brief, 7.5 105 MEC/tTAk cells (clone 29) were plated in 10-cm dishes, transfected with a plasmid mixture consisting of 10 g pTET-PA28, 10 g pTET-PA28, and 4 g pLXSP which confers resistance to puromycin, and then diluted in 96-well plates in medium containing 5 g/ml puromycin, 200 g/ml hygromycin B (Merck), and 400 ng/ml tetracycline (Sigma-Aldrich). Growing clones were screened for expression of PA28 and PA28 by immunoblot analysis using specific antisera. Isolation of Phen-DC3 PA28 Complexes. To isolate PA28 complexes, the pellets of 2 107 MEC-PA28 cells grown in the absence or presence of tetracycline for 3 d were lysed in 800 l of lysis buffer (0.1% Phen-DC3 Triton X-100, 50 mM Tris-HCl, 5 mM MgCl2, and 1 mM EDTA [pH 7.5]) without protease inhibitors. Cell lysates were freeze-thawed three times and then applied to a 10C40% glycerol gradient that was centrifuged for 16 h at 40,000 rpm in a Sorvall ultracentrifuge. Gradient fractions of 600 l were collected and tested for the presence of PA28 by Western blot analyses using PA28- and PA28-specific polyclonal rabbit antisera 10 27. To determine PA28 activity, 20 l of the glycerol gradient fractions and 80 l of assay buffer (50 mM Tris-HCl [pH 7.5], 25 mM KCl, 10 mM NaCl, 1 mM dithiothreitol, and 0.1 mM EDTA) containing 100 M Suc-LLVY-AMC were incubated in 96-well plates. To each well, Phen-DC3 30 ng of 20S proteasomes of nontransfected MECs were added and the reactions had been incubated for 1 h at 37C. Fluorescence emission was assessed at 460 nm (excitation 355 nm) using a Fluorostar audience. Isolation of Cellular 20S Proteasomes and Traditional western Blot Evaluation. Phen-DC3 Proteasomes had been purified from MEC217 cells cultured in the lack or existence of tetracycline (10 ng/ml and 400 ng/ml) as previously defined 26. Protein articles in the examples was quantified at an OD of 280 nm. 200 ng of materials was separated on 12% SDS polyacrylamide gels and electrophoretically used in nitrocellulose membranes. Blots had been incubated for 1 h in 10% equine serum/5% (wt/vol) lowfat dried out dairy/0.4% Tween-20 in PBS and probed overnight with polyclonal mouse LMP2-, -, MECL-1C, MC14-, LMP7-, MB1-, and MC3-particular rabbit antisera at a.