Expression of the fusion proteins was induced with isopropyl beta D thiogalactoside and the fusion proteins were extracted by lys ing the bacteria via sonication in a Triton X100 lysis buffer. After a high speed centrifugation to remove debris, the fusion protein containing supernatants were purified using glutathione conjugated agarose beads and the purified proteins were used to immunize mice selleck chemicals Belinostat for producing both polyclonal antisera and expressed as fusion proteins with a Red fluores cence protein fused to the N terminus. The recom binant plasmids were transfected into HeLa cells using the lipofectamine 2000 transfection reagent following the protocol recommended by the manufacture. The RFP chlamydial fusion proteins were visualized via either the fusion tag RFP or Inhibitors,Modulators,Libraries the mouse anti chlamydial protein antibody labeling 24 hours after transfection or as indcated in individual experiments.
To assess the effects of the RFP Cpn fusion proteins on the subsequent chlamy dial infection, the transfected cells were infected with C. pneumoniae AR39 organisms. The infected cultures grown on coverslips were processed for visualization of the trans fection and infection via an immunofluorescence assay. Cells expressing Inhibitors,Modulators,Libraries RFP were counted and % of RFP cells that contain chlamydial inclusions was calculated. In each experiment, 100 RFP cells Inhibitors,Modulators,Libraries were counted from 5 to 10 random views and three separate Inhibitors,Modulators,Libraries experiments were car ried out. The mean values were compared between the sample expressing RFP alone and samples expressing RFP Cpn fusion proteins using a two tailed Student t test.
The Inhibitors,Modulators,Libraries results were expressed as means plus minus standard errors. As a positive control, a recombinant pDsRed plas mid encoding the RFP GPICIncA fusion protein was sim ilarly transfected into HeLa cells followed by the C. caviae GPIC organism infection. The rates of GPIC infection in RFP cells were acquired and analyzed as described above. 4. Immunofluorescence assay HeLa cells grown on coverslips were fixed with 2% para formaldehyde dissolved in PBS for 30 min at room temperature, followed by permeabilization with 1% saponin for an additional 30 min. After washing and blocking, the cell samples were subjected to antibody and chemical staining. Hoechst was used to visualize nuclear DNA. A rabbit anti chlamydial organism antibody or anti CT395 plus a goat anti rabbit IgG secondary antibody conjugated with Cy2 was used to visualize chlamydial inclusions.
The mouse antibodies including both polyclonal antisera and monoclonal antibodies raised against various reference proteins and the C. pneumoniae GST fusion proteins plus a goat anti mouse IgG conjugated with Cy3 were used to visualize the corresponding antigens. In some cases, the primary antibodies were pre absorbed with either the cor responding or heterologous merely fusion proteins immobilized onto agarose beads prior to staining cell sam ples.