CsA and FK506 block Ca⁺⁺ ionophore–mediated inhibition of stimulus-induced up-regulation of LAT.

Jurkat T cells were pretreated with the indicated concentrations of FK506 (lanes 4-6) or CsA (lanes 7-9) for 1 hour at 37°C and then stimulated in the continuous presence of the inhibitors with the combinations of 1 ng/mL of PMA and 1 μM of Ca⁺⁺ ionophore A23187 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 3 times with similar results.