LAT expressed upon CD3 ligation and FK506 treatment distributes normally in the cell.
(A) A total of 2 × 107 Jurkat T cells in RPMI-5% FCS were incubated for 16 hours at 37°C in uncoated tissue culture dishes in the absence (A2) or presence (A3) of 5 ng/mL of FK506. For CD3 stimulation, Jurkat T cells in RPMI-5% FCS were incubated for 16 hours at 37°C in anti-CD3 mAb-coated tissue culture dishes in the absence (A4) or presence (A1 and A5) of 5 ng/mL of FK506. Following incubation, cells were removed from the dishes by gentle pipetting, fixed in suspension with 2% paraformaldehyde, and permeabilized with saponin. After permeabilization, the cells were incubated for 30 minutes at room temperature with 10 μg/mL of normal rabbit Ig (A1) or anti-LAT Ab (A2-A5), washed, and then incubated for 30 minutes at room temperature with goat-antirabbit IgG conjugated to Alexa 488. After incubation, the cells were washed in TBS/Tween and then analyzed by confocal scanning laser microscopy. Both immnuofluorescence (left panel) and corresponding differential interference contrast images (right panel) are shown. Original magnification, ×600. (B) Aliquots of the labeled cells in (A) were examined for LAT expression by flow cytometry. These experiments were repeated twice with similar results.
