Increase of cAMP inhibits TCR/CD3 plus CD28–mediated activation of ZAP-70 and phosphorylation of LAT.

Purified T cells were cultured either with media or with anti-CD3 plus anti-CD28 mAbs followed by cross-linking with rabbit antimouse immunoglobulin for 3 minutes. Where indicated, cells were pretreated with forskolin and IBMX. Cell lysates were prepared and immunoprecipitations were performed with ZAP-70–specific or LAT-specific antibody, followed by immunoblot with antiphosphotyrosine antibody. Immunoblots were stripped and sequentially reprobed with an antibody specific for ZAP-70.