The OMVs were also studied with regard to lipooligosaccharide (LOS) patterns using SDS-PAGE and silver staining of preparations treated with Proteinase K. The LOS was detected in the OMV samples and the pattern was identical to that of the whole cell samples (data not shown). The relative intensity of the major bands indicated that the LOS in the OMVs represented ca 0.2-0.5% of the total LOS of whole bacterial cells. Figure 3 Immunoblot detection of intra- and extra-cellular CDT of C. jejuni. Immunoblot
analyses of samples from C. jejuni wild type strains 81-176 (lanes 1-4) and the cdtA::km mutant (lanes 5-8). Samples: 1&5; whole cells (WC), JNK-IN-8 chemical structure 2&6; supernatants 1 (S1), 3&7; supernatants 2(S2), 4&8; OMVs, (A) Immunoblot detection with anti-CdtA polyclonal antiserum, (B) immunodetection with anti-CdtB polyclonal antiserum. (C) immunoblot detection with anti-CdtC polyclonal antiserum. (D) immunoblot detection with anti-Omp50 polyclonal antiserum.
Pictilisib nmr Immunoelectron microscopic analysis of proteis in OMVs To more directly monitor the association of CDT proteins with OMVs, we performed immunoelectron microscopic analyses. By immunolocalization using anti-CdtA, anti-CdtB, and anti-CdtC antibodies in the immunogold labeling method we detected the deposition of gold Wortmannin order particles on the vesicles obtained from CDT-producing bacteria (Figure 4A-C), whereas there was no labeling of OMVs from the CDT-negative strain (Figure 4D-F). We observed that some CDT containing vesicles were ruptured when the OMVs samples were mixed with antiserum in the immunogold experiment. The gold particles were mainly
observed on the material of the ruptured vesicles. It appeared that due to the rupture of the OMVs some of the released CDT subunits were accessible to the antiserum. The results strongly support the suggestion that the CDT proteins were indeed associated with OMVs of C. jejuni strain 81-176 and it appeared that the proteins might be internal or integral to the vesicle membrane. Since the C. jejuni Hsp60 protein that was somehow associated with OMVs as detected by SDS-PAGE analysis after the ultrcentrifugation step we also performed the immnunogold labelling and electron microscopic examination Reverse transcriptase using an Hsp60 recognizing polyclonal antiserum raised against the E. coli GroEL protein (Sigma-Aldrish). As shown in Figure 5B the gold particles labelled with anti-Hsp60 antiserum were observed not in direct association with OMVs but gold particles were associated with some amorphous material outside the OMVs. A similar immunogold labelling and analysis of the OMVs preparation with anti-Omp50 antiserum was shown in Figure 5C. In this case the gold particles were found to be localized in direct association with the OMVs as expected for an outer membrane protein. The results from these analyses indicated that the Hsp60 protein of C.