FIG. 3.

Overexpression of Tec, but not Bmx, Btk, or Itk, is sufficient to activate an NFAT reporter in Jurkat T cells. (A) Jurkat T cells were transfected with an NFAT-luciferase reporter construct, a β-galactosidase construct driven by the EF-1α promoter to control for transfection efficiency, and HA-tagged forms of Bmx, Btk, Itk, or Tec, with the quantities transfected indicated in micrograms. Sixteen hours posttransfection, cells were either left unstimulated or were stimulated with anti-TCR MAb C305 or PMA plus ionomycin. Six hours later, cells were lysed and assayed for luciferase and β-galactosidase, and expression of the Tec family constructs was confirmed by anti-HA Western blotting. Luciferase data were normalized for β-galactosidase values. Note the change of scale on the y axis of the PMA-ionomycin graph. The data are representative of at least three experiments. (B) Jurkat T cells were transfected with NFAT, AP-1, NF-κB, or RE-AP luciferase reporter constructs, in the presence or absence of 5 μg of HA-Tec. Luciferase assays were performed and are presented as described for panel A. Anti-CD28 MAb stimulation was included with anti-TCR stimulation for NF-κB and RE-AP assays.