The MEK inhibitor PD98059 blocks activation-induced up-regulation of LAT expression.

(A) Jurkat T cells were pretreated with the indicated concentrations of the MEK inhibitor PD98059 in RPMI-5% FCS (A and B, lanes 3-6) for 2 hours at 37°C and then stimulated in the continuous presence of the inhibitor with RPMI-5% FCS containing 1 μg/mL of anti-CD3 mAb (A) or RPMI-5% FCS containing 1 ng/mL of PMA (B) for 16 hours at 37°C. After incubation, the cells were immediately lysed with boiling 2× SDS-PAGE sample buffer. Proteins in WCL were separated by SDS-PAGE and then transferred to membranes and immunoblotted with anti-LAT mAb or anti-Csk Ab. This experiment was repeated 4 times with similar results. (C) Inhibition of Erk phosphorylation by PD98059. Jurkat T cells were incubated in RPMI-5% FCS (lanes 1 and 2) or RPMI-5% FCS containing 200 μM of PD98059 (lane 3) for 16 hours at 37°C. After incubation, the cells were washed with RPMI-0.001% bovine serum albumin and then suspended in the same media. The cells were then left untreated (lane 1) or were stimulated with 1 μg/mL of anti-CD3 mAb for 15 minutes (lanes 2 and 3). After incubation, the cells were immediately lysed by adding an equal volume of 2× boiling SDS-PAGE sample buffer, and the proteins in WCL were separated by SDS-PAGE and then transferred to membranes and immunoblotted with anti-phospho-Erk mAb. This experiment was repeated 3 times with similar results.