Ligation of TCR or CD28 or PMA treatment up-regulates the expression of LAT in T cells.

Purified human normal resting T cells (A) or Jurkat T cells (B) were incubated for 16 hours at 37°C in 96-well tissue culture plates in RPMI-5% FCS (lane 1) or in RPMI-5% FCS containing the indicated concentrations of anti-CD3 mAb (lanes 2-5). After incubation, the cells were immediately lysed with 2× boiling SDS-PAGE sample buffer. After boiling, proteins in WCL were separated by SDS-PAGE and then transferred to membranes and immunoblotted with anti-LAT mAb (upper panel) or anti-Csk Ab (lower panel). (C) Jurkat T cells were incubated as described above for 16 hours at 37°C in RPMI-5% FCS (lane 1) or in RPMI-5% FCS containing the indicated concentrations of anti-CD28 mAb (lanes 2-5). Proteins were processed as described above. (D) Jurkat T cells were incubated as described above for 16 hours at 37°C in RPMI-5% FCS (lane 1), RPMI-5% FCS containing the indicated concentrations of PMA (lanes 2-4), or RPMI-5% FCS containing 4α-PMA (lanes 5-7). Proteins were processed as described above. (E) Purified human normal resting T cells were incubated as described above for 16 hours at 37°C in RPMI-5% FCS (lane 1) or RPMI-5% FCS containing the indicated concentrations of PMA (lanes 2-4). Proteins were processed as described above. (F) Jurkat T cells were incubated as described above for 16 hours at 37°C in RPMI-5% FCS (lane 1) or RPMI-5% FCS containing the indicated concentrations of the Ca⁺⁺ ionophore A23187 (lanes 2-5). Proteins were processed as described above. Each of the experiments in this figure was performed at least 3 times with similar results.