Journal: Microbiology

Article Title: The trans-Golgi SNARE syntaxin 6 is recruited to the chlamydial inclusion membrane

doi: 10.1099/mic.0.045856-0

Figure Lengend Snippet: Localization of endogenous syntaxin proteins to the chlamydial inclusion. (a) C2BBe1 cells were seeded onto glass coverslips for 48 h prior to infection with C. trachomatis serovar L2 (m.o.i. 6 : 1) for an additional 18 h. Cells were fixed in absolute ethanol for 30 min at −20 °C and processed essentially as described in Methods. Samples were visualized with an LSM 510 Laser Module Zeiss Axiovert 200M confocal microscope. Arrows indicate chlamydial inclusions. (b) C2BBe1 cells were transfected with eGFP-syntaxin 6 and infected with C. trachomatis L2 for 18 h. Cells were fixed with methanol and counterstained with an anti-EB antiserum. Bars, 10 μm.

Article Snippet: Samples were then processed for indirect immunofluorescence using rabbit anti-IncG (inclusion membrane protein), mouse anti-syntaxin 4 (BD Biosciences), mouse anti-syntaxin 16 (Synaptic Systems) or mouse anti-syntaxin 6 (BD Biosciences).

Techniques: Infection, Microscopy, Transfection

Journal: Microbiology

Article Title: The trans-Golgi SNARE syntaxin 6 is recruited to the chlamydial inclusion membrane

doi: 10.1099/mic.0.045856-0

Figure Lengend Snippet: Localization of syntaxin 6 to the inclusions of multiple chlamydial species. C2BBe1 cells were seeded onto coverslips and transfected with mCherry-syntaxin 6 or with eGFP-syntaxin 6, then infected with C. trachomatis serovar L2 (m.o.i. 6 : 1), C. muridarum (m.o.i. 0.1 : 1), C. caviae (m.o.i. 0.2 : 1), C. pneumoniae (m.o.i. 13 : 1) or Coxiella burnetii Nile Mile phase II (m.o.i. 50 : 1) as described in Methods. To terminate the infections, cells were fixed in methanol and processed for indirect immunofluorescence to detect the organisms (green). Samples were visualized with an LSM 510 Laser Module Zeiss Axiovert 200M confocal microscope. Bar, 10 μm.

Article Snippet: Samples were then processed for indirect immunofluorescence using rabbit anti-IncG (inclusion membrane protein), mouse anti-syntaxin 4 (BD Biosciences), mouse anti-syntaxin 16 (Synaptic Systems) or mouse anti-syntaxin 6 (BD Biosciences).

Techniques: Transfection, Infection, Immunofluorescence, Microscopy

Journal: Microbiology

Article Title: The trans-Golgi SNARE syntaxin 6 is recruited to the chlamydial inclusion membrane

doi: 10.1099/mic.0.045856-0

Figure Lengend Snippet: Chlamydial protein synthesis requirement for syntaxin 6 localization to the inclusion. C2BBe1 cells were seeded onto glass coverslips in 24-well plates 48 h prior to infection with C. trachomatis serovar L2 (m.o.i. 9 : 1). After 18 h, cells were either fixed in absolute ethanol (−Chlor) or treated with 200 μg ml −1 for an additional 24 h (+Chlor), then fixed in absolute ethanol and processed for indirect immunofluorescence essentially as described in Methods. IncG staining was used to identify the inclusion. Samples were visualized with an LSM 510 Laser Module Zeiss Axiovert 200M confocal microscope. Bar, 10 μm.

Article Snippet: Samples were then processed for indirect immunofluorescence using rabbit anti-IncG (inclusion membrane protein), mouse anti-syntaxin 4 (BD Biosciences), mouse anti-syntaxin 16 (Synaptic Systems) or mouse anti-syntaxin 6 (BD Biosciences).

Techniques: Infection, Immunofluorescence, Staining, Microscopy

Journal: Microbiology

Article Title: The trans-Golgi SNARE syntaxin 6 is recruited to the chlamydial inclusion membrane

doi: 10.1099/mic.0.045856-0

Figure Lengend Snippet: Identification of the syntaxin 6 protein domain mediating localization to the chlamydial inclusion. (a) Functional domains of syntaxin 6. These include the 24 amino acid H1 domain, the 10 amino acid plasma membrane retrieval signal (PMRS), including the YGRL tyrosine motif and the 59 amino acid H2 domain. Also depicted is the C-terminal transmembrane domain (TM); this domain anchors syntaxin 6 in vesicular membranes . (b) Examination of the involvement of syntaxin 6 functional domains in localization to the chlamydial inclusion. C2BBe1 cells were transfected with the indicated 3XFLAG-syntaxin 6 (syn6) constructs, followed by infection with C. trachomatis serovar L2 (m.o.i. 4 : 1). At 18 h post-infection, cells were fixed in absolute ethanol and processed for indirect immunofluorescence. Chlamydial inclusions were labelled with an antibody that recognizes the inclusion membrane (IncG), and the syntaxin 6 constructs were detected with an anti-M2 FLAG tag antibody (Sigma-Aldrich). Slides were visualized with an LSM 510 Laser Module Zeiss Axiovert 200M confocal microscope. Bar, 10 μm.

Article Snippet: Samples were then processed for indirect immunofluorescence using rabbit anti-IncG (inclusion membrane protein), mouse anti-syntaxin 4 (BD Biosciences), mouse anti-syntaxin 16 (Synaptic Systems) or mouse anti-syntaxin 6 (BD Biosciences).

Techniques: Functional Assay, Transfection, Construct, Infection, Immunofluorescence, FLAG-tag, Microscopy