Cells were left for 10 min on poly-lysine coated slides (Thermo Scientific) prior fixation with PBS1X containing 4% paraformaldehyde. For TCR crosslinking experiments, cells were incubated with 5 mg.ml 21 anti-CD3 at 4uC for 15 min. After two washes, cells were incubated with 5 mg.ml 21 of goat antimouse (Jackson) for 20 min either at 4uC or 37uC. To disassemble Golgi apparatus, cells were treated with 10 mg.ml 21 Brefeldin A for 60 min. Samples were permeabilized with 0.05% Triton-X100 in PBS1X for 5 min, and non-specific sites blocked with 10% FCS in PBS1X. Antibodies used were: PALS1 (Millipore), 58K Golgi (Abcam), Alexa-488 conjugated goat anti-rabbit IgG or Alexa-594 conjugated goat anti-mouse IgG (Invitrogen). Samples were analyzed using a Leica confocal microscope SP6.