Figure 3

Hck-Src homology 2 (SH2) domain interacts with the phosphorylated immunoreceptor tyrosine-based activation motif (ITAM)-like sequence of carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3) in intact cells. 293T cells were cotransfected to express CEACAM3 wild type tagged with cyan fluorescent protein (CEACAM3 WT-CyPet) or CEACAM3 without the cytoplasmic domain (CEACAM3 ΔCT-CyPet), together with enhanced yellow fluorescent protein (YPet)-Hck-SH2 and v-Src as indicated. At 2 days later, cells were analyzed by flow cytometry. (a) Cell populations expressing donor or acceptor constructs alone or cotransfected with CyPet-encoding and YPet-encoding constructs were identified. Dot plots show the CyPet and YPet fluorescence of the indicated samples. Squares indicate the population of CyPet and YPet double-positive cells coexpressing CEACAM3 WT and YPet-Hck-SH2. (b) Histogram of fluorescence intensity in the fluorescence resonance energy transfer (FRET) channel of gated CyPet and YPet double-positive cells as shown in (a). (c) Mean fluorescence intensity in the FRET channel of gated CyPet and YPet double-positive cells as shown in (a).