Fig. 2.

HER2-CRB induces NK cell immunological synapse formation and signaling. A Diagram of HER2-CRB, NIST-CRB and HER2/NIST molecules. B Flow cytometry-based conjugate quantification following 10 min incubation of primary NK cells conjugated with BT474 cells in the absence or presence of 1 or 10 µg/mL HER2-CRB. Results obtained from two independent healthy donors. Representative dot plot (left) and quantified data (right). C Representative images of cell trace violet (CTV)-labeled primary NK cells conjugated with CMTMR-labeled BT474 cells in the presence of NIST- or HER2-CRB. Quantified area depicting immunological synapse used to quantify phosphotyrosine (pTyr) and phalloidin signals. D Quantification of actin and pTyr intensities by confocal imaging of 30 individual interactions after 10 min incubation of conjugation of primary NK cells with BT474 cells in the presence of NIST- or HER2-CRB. Similar results were obtained from another independent healthy donor. E Western blot of phosphorylated ERK, total ERK and loading control Hsp60 after 5 and 15 min incubation of NK cells, HER2-coated beads and either HER2-CRB or NIST-CRB. Mann–Whitney test used for statistics; ****, p ≤ 0.0001