Figure 1.

Syntaxin 10 localization to the chlamydial inclusion. (A) HeLa cells were transfected with 3XFLAG-syntaxin 10 (3XF-stx10) for 24 h prior to fixation and processing for imaging. The Golgi (outlined in white) was detected with a rabbit anti-giantin antibody; 3XF-Stx10 was detected using a mouse anti-FLAG M2 antibody. (B,C) HeLa or HEp2 cells transfected with 3XF-stx10 were infected for 16–18 h with C. trachomatis serovar L2, prior to fixation and processing for imaging. The inclusion membrane was detected using either a rabbit anti- IncA (B) (Additional images provided in Supplemental Figure 1) or IncG (C) antibody; 3XF-Stx10 was detected as above. To distinguish 3XF-Stx10 from surrounding cell structures, some samples were treated with brefeldin A (BFA) to collapse the surrounding Golgi. To examine if chlamydial protein synthesis was required for 3XF-Stx10 localization, infected monolayers were treated with 200 μg/ml chloramphenicol (Chlor) for an additional 24 h prior to fixation. In chloramphenicol treated cells, white asterisks indicate inclusions. All images were acquired using an Olympus Fluoview 1000 Laser Scanning Confocal Microscope with a 60X objective and 2x zoon. These results are representative of at least 3 independent experiments. White bars = 10 μm.