Lck and ZAP-70 are both necessary for T-cell migration on ICAM-1. (A) T lymphoblasts were either untreated or treated with 40μM Src family inhibitor PP2 or control PP3 and incubated on immobilized ICAM-1 before observation for 20 minutes by video microscopy and the migration of individual cells tracked. A boxplot (box-and-whisker plot) of 3 independent migration experiments is shown in the top panel and a representative experiment showing the migratory tracks of individual cells from each treatment condition in the bottom panel; n = 40 cells per condition. (B) T lymphoblasts were either untreated or treated with 10μM Syk family inhibitor piceatannol and processed as in panel A. (C) HSB2 T cells were electroporated without siRNA as a control, with 400nM control siRNA or ZAP-70 siRNA. Western blots were probed for total ZAP-70 and α-tubulin served as a loading control. (D) HSB2 T cells that were either untreated, electroporated without siRNA, with control siRNA or with ZAP-70 siRNA were observed on ICAM-1 by video microscopy for 20 minutes. A boxplot of 3 independent migration experiments is shown. (E) Confocal images of typical HSB2 T cells transfected with control siRNA or with ZAP-70 siRNA on ICAM-1. The T cells are stained for actin with phalloidin–Alexa 546. Quantification of the proportion of polarized T cells after siRNA transfection: 74%, control; 27%, ZAP-70 knockdown; n = 105 cells for each condition.