Figure 2.

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). 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 after transfection cells were lysed. (a) Western blot of the whole cell lysates (WCL) with anti-hemagglutinin (HA) antibodies (upper panel) or anti-green fluorescent protein (GFP) antibodies (lower panel) confirmed expression of the indicated proteins after transfection. (b) CEACAM3 WT was immunoprecipitated from lysates and analyzed by western blotting with anti-phosphotyrosine antibodies. (c) Fluorescence of lysates harboring the indicated proteins was determined in three separate channels and apparent fluorescence resonance energy transfer (FRET) efficiency (E_Aapp; black bars) was calculated as described. Purified glutathione S-transferase (GST)-Src-SH2 was added as a competitive inhibitor of YPet-Hck-SH2 to the lysates and measurements were repeated (open bars). (d) Purified GST, GST-Src-SH2, or buffer were added to lysates of cells expressing CEACAM3 WT-CyPet, YPet-Hck SH2 and v-Src. FRET efficiency (E_Aapp) was calculated as in (c). Bars represent mean values ± standard error of the mean (SEM) of three independent experiments.