FIG 6.

Stx6 is localized on EEA1+ vesicles rather than the TGN in primary monocytes. Three distinct primary antibodies targeting Stx6 were used to assess the localization of Stx6 in uninfected primary human monocytes. (A) Anti-Stx6 targeting amino acids 1 to 255. (B) Anti-Stx6 targeting amino acids 50 to 200. (C) Anti-Stx6 targeting amino acids 1 to 25. Super resolution microscopy was performed as described in Fig. 4 (D–F). Uninfected primary human monocytes were fixed and stained with antibodies to EEA1 (A) or TGN46 (B) and Stx6. High resolution images were acquired on a Nikon N-SIM E Super Resolution microscope system (100× objective) in single slices (z stacks) using the same laser and lookup table (LUT) settings. Representative images are shown. Stx6 colocalization (~0.1 μm distance) with target organelle was manually quantified using the companion analysis software of the microscope (F). HCMV TB40/E (MOI = 10) -infected primary human monocytes were fixed at 15 mpi (G) and 2 hpi (H) and stained with antibodies against EEA1 or TGN46, HCMV UL32-GFP, and Stx6. High resolution images were acquired on a Nikon N-SIM E Super Resolution microscope system (100× objective) in single slices (z stacks) using the same laser and LUT settings. Representative images are shown. White arrows indicate co-localization between organelle and Stx6. N = 3 for all experiments.