Ca⁺⁺ ionophores completely block activation-induced up-regulation of LAT expression.

Jurkat T cells were incubated with RPMI-5% FCS containing 1μg/mL of anti-CD3 mAb (A) or RPMI-5% FCS containing 1 ng/mL of PMA (B) in the absence (A and B, lane 2) or presence of the indicated concentrations of the Ca⁺⁺ ionophores A23187 (A and B, lanes 3-5) or ionomycin (Iono; A and B, lanes 6-8) 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. This experiment was repeated 3 times with similar results. (C) Jurkat T cells were incubated with RPMI-5% FCS containing 1 ng/mL of PMA in the presence of the indicated concentrations of Ca⁺⁺ ionophore A23187 or ionomycin (Iono) for 16 hours at 37°C. After incubation, the tubes were centrifuged and the supernatants were collected and analyzed for IL-2. (D) Purified human normal resting T cells were incubated with RPMI-5% FCS (lane 1) or RPMI-5% FCS containing 1 ng/mL of PMA in the absence (lane 2) or presence of the indicated concentrations of the Ca⁺⁺ ionophore A23187 (lanes 3-5). (E) Jurkat T cells were incubated for 16 hours at 37°C in RPMI-5% FCS (lane 1) or RPMI-5% FCS containing 1 ng/mL of PMA (lane 2-5). After incubation, PMA-pretreated cells were washed and then incubated for 8 hours at 37°C with RPMI-5% FCS (lane 3), RPMI-5% FCS containing 1 ng/mL of PMA (lane 4), or RPMI-5% FCS containing 1 μM of Ca⁺⁺ ionophore A23187 (lane 5). This experiment was repeated twice with similar results.