FIG. 4.

Detection of trimeric Pgp3 in chlamydial outer membrane complexes. (A) Lysates of C. trachomatis serovar D-infected HeLa cells (lane 1), purified EBs (lane 2), or insoluble fractions from extraction of purified EBs with Sarkosyl for the number of times indicated on top of the figure (lanes 3 to 6) were subjected to electrophoresis in a 12% denaturing gel. The resolved proteins were detected by Western blot (WB) analysis of Pgp3 (panel a), MOMP (panel b), HSP60 (panel c), and CPAF (panel d) with the corresponding antibodies as listed along the left side of the figure. Note that although the chlamydial cytosolic protein HSP60 was efficiently removed from the insoluble fraction by Sarkosyl extraction, both MOMP and Pgp3 were still associated with the pellet even after six cycles of extraction with Sarkosyl, indicating that Pgp3 is a stable component of the chlamydial outer membrane complex. CPAFc, C-terminal half of CPAF. (B) An insoluble fraction generated by six cycles of Sarkosyl extraction was further extracted with no additives (lanes 1 and 2) or with Sarkosyl (lanes 3 and 4) or 2% SDS (lanes 5 and 6) in the presence (lanes 2, 4, and 6) or absence (lanes 1, 3, and 5) of DTT. The corresponding supernatants were loaded to a 12% native gel for monitoring levels of both Pgp3 and MOMP by Western blot analysis. Note that Sarkosyl treatment in the presence of DTT released significant amounts of both Pgp3 trimers (lane 3 versus lane 4 in panel a) and MOMP (b). Extraction with SDS denatured Pgp3 into monomers with DTT (panel a, lane 6) or without DTT (lane 5). The presence of DTT plus SDS resulted in the conversion of MOMP oligomers into monomers (lane 5 versus lane 6 in panel b).