Figure 2.

LFA-1 dependent responses are ablated by inhibition of the Syk kinase. (A) NK cells pretreated for 45 minutes at 37°C in culture medium with 1 μM Syk inhibitor II or DMSO carrier were mixed with Celltracker Green labeled S2 cells or S2 cells expressing ICAM-1 for 20 min at 37°C, adhered to poly-D-lysine coated slides, fixed, permeablized, and stained with an anti-perforin antibody and Alexa-568 goat-anti-mouse secondary antibody. A representative image of treated or untreated NK cells in contact with S2-ICAM-1 cells is shown. (B) Treated or untreated NK cells in conjugate with S2 or S2-ICAM-1 cells were scored for polarization of perforin toward the target cell interface. Error bars represent S.D. of three experiments conducted with NK cells from different donors. *p < 0.05 relative to DMSO-treated NK cells + S2-ICAM-1 cells. (C) NK cells were pretreated for 45 minutes at 37°C in culture medium with 1μM Syk inhibitor II (Calbiochem) or DMSO carrier. Cells were then stimulated with purified ICAM-1 on plates for 20 minutes at 37°C and lysed. Pyk2, CasL, and paxillin immunoprecipitations and whole cell lysates (WCL) were resolved by SDS-PAGE and immunoblotted for phosphotyrosine.