FIG. 4.

Sin expression inhibits TCR-induced transcriptional activation. Jurkat Tag cells were transiently transfected with increasing amounts of plasmid DNA expressing Sin (A and D), CasL (B), or SLP-76 (C) in the presence of NFAT- or AP-1-firefly-luciferase (luc) as shown along with the HSV-TK-Renilla-luciferase reporter (A to E). The fold activation for NFAT-and AP-1-firefly-luciferase and actual luciferase units for HSV-TK-Renilla-luciferase are shown (A, bottom graph). (E) Jurkat Tag cells were transfected with a pEBB vector (vec) expressing Sin, SinII/Efs2, or SinΔC (300 ng each) in the presence of the NFAT- and HSV-TK-luciferase reporters as shown. The results represent one of at least three experiments each performed in triplicate, and fold activation is relative to the value obtained with pEBB vector backbone used to express Sin, CasL, or SLP-76 in unstimulated cells, which was given a value of 1. The results shown represent the mean ± SD. Whole-cell lysates of the transfected cells were separated on SDS-PAGE and Western blotted with anti-Sin (α-Sin) or anti-p130Cas (α-p130Cas) antibodies to reveal levels of Sin and CasL protein expression, respectively (A, B, and E, insets).