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. 2010 Sep 17;192(22):6017–6024. doi: 10.1128/JB.00847-10

FIG. 3.

FIG. 3.

Detection of Pgp3 trimers in chlamydia-infected cell samples. (A) HeLa cells infected with C. trachomatis were processed for antibody labeling in an immunofluorescence assay. CPAF and Pgp3 were labeled with MAbs 100a and 2H4, respectively (red), and the chlamydial organisms were labeled with a rabbit antibody (green) and DNA with Hoechst dye (blue). Note that MAb 2H4, known to recognize only native trimeric Pgp3, detected signals in both inclusions (white star) and cytoplasms (white arrows) of chlamydia-infected cells, suggesting that the Pgp3 in the chlamydia-infected cells also represented trimers. (B) Lysates of HeLa cells alone (lane 1) or HeLa cells with C. trachomatis serovar D infection (D-HeLa; lane 2), cytosolic fractions of D-HeLa cells (S100; lane 3), and purified EB extracts (lane 4) were loaded to native gradient (panel a) or denaturing (panels b to d) gels for Western blot analyses with a mouse anti-Pgp3 polyclonal antibody (pAb; panels a and b), an anti-CPAF MAb (100a; panel c), or a rabbit anti-chlamydial major outer membrane protein (MOMP) polyclonal antibody (pane d) as indicated on the left of the figure. Note that the Pgp3 that both secreted into the host cytosol and associated with the purified EBs was in the form of trimers. CPAFc, C-terminal half of CPAF.