Overview of the used constructs. (a) Wild type carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3 WT) comprising an immunoreceptor tyrosine-based activation motif (ITAM)-like sequence in its cytoplasmic domain (CT) or CEACAM3 ΔCT lacking the entire cytoplasmic domain were C-terminally tagged with cyan fluorescent protein (CyPet), as a fluorescence resonance energy transfer (FRET) donor, or enhanced green fluorescent protein (EGFP), respectively. Two specific tyrosine residues within the ITAM-like sequence are known to be phosphorylated upon receptor activation (indicated by asterisks). Hck-Src homology 2 (SH2) domain was cloned as a 3' fusion to far-red fluorescent protein (mKate) or enhanced yellow fluorescent protein (YPet), which served as FRET acceptor. (b) The SH2 domain of Hck is recruited to CEACAM3, when cells are infected with OpaCEA-expressing Neisseria gonorrhoeae. 293T cells were cotransfected with expression plasmids encoding the indicated GFP-tagged CEACAM3 variants and mKate-Hck-SH2 as indicated. Cells were infected with AF647-labeled OpaCEA-expressing N. gonorrhoeae at a multiplicity of infection of 30 bacteria/cell. Scale bar corresponds to 10 μm.