Specific inhibition of Src kinase activity prevents lytic granule convergence to the MTOC. Fixed cell confocal microscopy of (A) eNK cells pretreated with DMSO (vehicle control), PP2, PD 98059, or AZM 475271 and (B) YTS cells pretreated with DMSO or AZM 475271 for 30 minutes at 37°C before activation on an anti-NKp30/CD18–coated surface for eNK cells or anti-CD28/CD11a–coated surface for YTS cells for 15 minutes at 37°C, followed by 10 minutes of fixation at room temperature. In each image, confocal immunofluorescence in the plane of the MTOC is shown. Green, anti–α-tubulin detected by Pacific Blue-streptavidin; red, fluorescein isothiocyanate anti-perforin. Quantitative analyses of lytic granule distance from the MTOC are shown as a feature of inhibitor treatments in (C) eNK cells and (D) YTS cells. Data represent >20 cells per condition from independent experiments using 3 healthy donors for eNK cells and 3 independent experiments for YTS cells. Error bars show ±SD. Mean distance of lytic granules from the MTOC was significantly larger in eNK cells after PP2 and AZM 475271 (***P < .001) and in YTS cells after AZM 475271 II treatment (***P < .001). (E) Western blot analysis of YTS cells incubated on an anti-CD56–coated surface (left lane) or anti-CD28/CD11a–coated surfaces (right lanes). For the rightmost lanes, cells were pretreated with Src kinase inhibitors PP2 or AZM475271. The blot is representative of 4 independent repeats. (F) Quantitative analysis of the blot shown in panel E with pSrc values normalized to those of Myosin IIA, which was included as a loading control.