Fig. 2. Tyrosine phosphorylation and raft partition of LAT constructs. (A) LATWT-, LAT32-104- and LATCt-GFP cells were left unstimulated or triggered with anti-CD3 mAb for 2 minutes, and were then lysed and immunoprecipitated with anti-GFP. Immune complexes were sequentially analysed by anti-phosphotyrosine and anti-GFP immunoblotting. (B) Postnuclear supernatants obtained from sonicated LATWT- and LATCt-GFP cells were lysed in Brij 98 at 37°C followed by sucrose gradient fractionation. Raft (L) and non-raft fractions (H) were collected and analysed by anti-GFP immunoblotting. (C) LATWT-, LAT32-104- and LATCt-GFP cells were transfected with the red-shifted GFP (RSGFP) construct. After 36 hours, cells were incubated or not in PBS 1%, Triton X-100 for 15 minutes at 4°C and mounted on slides. RSGFP and GFP staining was analysed by confocal microscopy.