Fig. 4. Tyrosine residues 136, 175, 195 and 235 are required for TCR-induced recruitment of LAT to the immune synapse. (A) Schematic representation of the mouse LAT and LATY/F GFP chimaeras. (B) LATWT- and LATY/F-GFP cells were left unstimulated or triggered with anti-CD3 mAb for 2 minutes. Whole cell lysates (WCL) and GFP immunoprecipitates (IP) were sequentially analysed by anti-pTyr and anti-GFP immunoblotting. (C) LATWT- and LATY/F-GFP cells were mixed with CellTracker^TM Orange-labelled APCs loaded for 20 minutes with 5 µg/ml SEE and monitored by time-lapse video microscopy. Images were taken at 15-second intervals. Representative images from the digital movies at the indicated times (seconds) are shown. White arrowheads point to the site of initial contact.