The Hck Src homology 2 (SH2) domain directly associates with carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3) at sites of bacterial contact. (a) 293T cells were cotransfected with expression plasmids for CEACAM3 wild type tagged with cyan fluorescent protein (WT-CyPet) and enhanced yellow fluorescent protein (YPet)-Hck-SH2. At 2 days later, cells were infected with AF647-labeled OpaCEA-expressing Neisseria gonorrhoeae for 30 min and fixed. The cell was imaged before and after photobleaching of the acceptor in a defined region, where bacteria were bound to CEACAM3. Scale bar: 10 μm. (b) Enlargement of the marked region in (a) in pseudocolor including the acceptor-bleached area. Fluorescence resonance energy transfer (FRET) was calculated as described in Methods. Scale bar corresponds to 5 μm.