Journal: Methods in Molecular Biology

Article Title: Chlamydia trachomatis

doi: 10.1007/978-1-4939-9694-0

Figure Lengend Snippet: Fig. 1 Confirmation of BioID and APEX2 specific biotinylation using indirect immunofluorescence assays and western blotting. (a) HeLa cells transfected with Syntaxin10-BirA∗were mock-infected or infected with C. trachomatis L2 in media supplemented with 50 μM biotin to catalyze the labeling of proximal proteins with biotin. Cells were fixed at 23 h postinfection and processed for immunofluorescence to visualize the inclusion membrane in infected cells (anti-IncA, pink) or the Golgi in uninfected cells (anti-Giantin, pink), expression of Syntaxin10-BirA∗(anti-Myc, red), biotinylated proteins (streptavidin–488 conjugate, green) and DNA (DAPI, blue). Images were taken at 100 magnification using a Zeiss Apotome 2.1. Scale bar ¼ 10 μm. (b) HeLa cells infected with C. trachomatis L2 transformed with IncA-APEX2 or APEX2 only and induced with anhydrotetracycline (aTc) at 7 hpi. Biotin-phenol was added 30 min prior to the biotin labeling step at 24 hpi. Biotinylation was catalyzed by the addition of 3 mM H2O2 for 1 min and washed with a quenching solution. Coverslips were fixed and processed for immunofluorescence to visualize the inclusion membrane (anti-CT223, pink), expression of the construct (anti-Flag, red), biotinylated proteins (streptavidin–488

Article Snippet: Fluorescent streptavidin conjugate (e.g., Streptavidin–488 Jackson ImmunoResearch Laboratories Inc., 016-540-084).

Techniques: Western Blot, Transfection, Infection, Labeling, Membrane, Expressing, Transformation Assay, Construct