The processing and MHC class I-restricted presentation of antigenic peptides derived from the p60 protein of the facultative intracellular bacterium is tightly linked to bacterial protein synthesis. viral pathogens, bacteria provide their own protein biosynthesis apparatus. It is not known if in analogy to cellular RDP inside cells also a portion of bacterial proteins is rapidly degraded. In the current study the accumulation and decay of p60 in p60-derived antigenic peptides p60 217C225 and in p60 449C457 are generated with much higher efficacies of 1 1 antigenic peptide per 35 and 1 per 1.7 degraded p60 molecules, respectively [11], [12], [13], [16]. Thus in contrast to viral antigenic peptides a very high percentage of an antigen has to be degraded in order to generate sufficient antigenic peptides for antigen presentation. Both, p60 degradation as well as antigen presentation of p60 217C225 and p60 449C457 are inhibited by proteasome inhibitors [17], [18]. Also after inhibition of bacterial protein synthesis the pool of accumulated p60 molecules is usually inhibited by proteasome inhibitors, thus linking the degradation of this p60 pool to the proteasome. An important requirement for our experimental approach for the detection of putative p60-RDP is the inhibition Apixaban of p60 degradation without SERP2 inhibiting bacterial replication or bacterial protein synthesis. Inhibition of bacterial protein synthesis would result in lower protein levels and such Apixaban strongly influence the calculation of degraded antigens. We used epoxomicin for all those proteasome inhibition experiments because it has the least effect on bacterial replication and p60 synthesis among a panel of various proteasome inhibitors [17]. It is currently unknown, however, why the proteasome-mediated degradation of accumulated p60 molecules does not yield antigenic peptides that are presented in the context of MHC class I molecules [11], [12]. Without the tight linkage to bacterial protein synthesis the presentation of p60-derived antigens should continue after antibiotic inhibition of protein synthesis because a few hours after contamination the accumulated p60 molecules could provide antigenic peptides for several hours [13]. It has previously been shown that intracellular are recognized by the ubiquitin system resulting in spatial association of proteasomes and intracellular bacteria [19], providing a possible direct link between bacteria, bacterial protein translation, and antigen presentation. and the processing of p60-derived antigenic peptides is usually impartial of putative p60-RDPs. Materials and Methods Bacteria and cell Apixaban lines All in vitro infections were performed with 43251 [12]. Bacteria were produced at 37C in brain heart infusion bouillon (BD, Heidelberg, Germany) and used for contamination in the logarithmic growth phase. The bacterial concentration was estimated from your OD at 600 nm. The macrophage-like J774A.1 (J774) cell collection was purchased from your American Type Culture Collection (Manassas, VA) and was grown in DMEM (Invitrogen, Karlsruhe, Germany) cell growth medium containing 10% FCS (PAA, Pasching, Austria). Reagents The irreversible proteasome inhibitor epoxomicin (SIGMA, Deisenhofen, Germany) was used at concentrations of 2 M. Epoxomicin was dissolved in dimethyl sulfoxide to yield a 1000 stock solution and stored at ?80C until use. Total protease inhibitor cocktail tablets were from Roche (Mannheim, Germany) und used according to the suggestions of the manufacturer. DNAse and RNAse were purchased from SIGMA (Deisenhofen, Germany). Tetracyclin (SIGMA) was dissolved in methanol to yield a 500 stock answer (10 mg/ml) stored at ?20C until use. Quantitative western blot analysis J774 cells were produced to 70% confluence in 6 cm cell culture dishes in 4 ml DMEM supplemented with 10% FCS without antibiotics. Cells were infected for 30 min with 0.4 ml of a log phase culture of (OD600?=?0.1). Infected cells were washed twice with DMEM supplemented with 10% FCS and cultured in DMEM with 10% FCS made up of 15 g/ml gentamicin to inhibit extracellular bacterial growth. Epoxomicin was added 30 min after contamination to yield final concentrations of 250 M and 2 M, respectively. Tetracycline was added to a final concentration of 20 g/ml. At indicated time points cells were washed twice with PBS and lysed in 300 l of lysis buffer (1 TRIS-buffered saline, 0.1% Triton X-100, Complete protease inhibitor, 50 U/ml DNAse, 50 U/ml RNAse). After 15 min at room temperature lysates were cleared by centrifugation at 14.000 g for 10 min. Cell lysates were prepared by heating in SDS-PAGE sample buffer (BioRad, Mnchen, Germany) for 5 min. Proteins were separated by SDS-PAGE (10% gel) and blotted onto nitrocellulose (Schleicher & Schuell BioSciences, Dassel, Germany). Blots were blocked in 5% (w/v) dry milk and probed with a 11.000 dilution of a polyclonal rabbit anti-antibody (batch R2556) for 4 h and simultaneously probed with rabbit anti-GAPDH and goat anti-GAPDH (abcam, Cambridge, UK) as.