<t>TAB1</t> inhibits IL-2 and increases IL-10 production. (A) T-cell hybridoma 2B4 was transduced with retrovirus that contained the vector alone (Mock) or human TAB1 (TAB1), and stable cell lines were selected in the presence of G418 (500 μg/ml). RNA was prepared from each cell line and was subjected to RT-PCR analysis for TAB1 mRNA expression. PCR was performed for 18, 24, 30, and 36 cycles (as indicated by the triangles over the lanes). (B) p38α was immunoprecipitated with specific antibodies from lysates of Mock-2B4 cells (Mock) or TAB1-2B4 cells (TAB1) and was subjected to an in vitro kinase assay with ATF2 19-96 peptide as a substrate. The quantity of p38α was assessed by immunoblotting with anti-p38α antibody. (C) Mock-2B4 cells (Mock) and TAB1-2B4 cells (TAB1) were incubated with (SB) or without (Nil) SB203580 (10 μM) for 2 h, lysed, and subjected to immunoblotting with anti-phospho-p38 and anti-p38α antibodies. (D) Mock-2B4 cells (Mock) and TAB1-2B4 cells (TAB1) were cultured for 18 h in plates coated (TCR) or not coated (Nil) with anti-TCR MAb (10 μg/ml) and were subjected to RT-PCR analysis for IL-2, IL-10, and G3PDH mRNAs. (E) Mock-2B4 cells (Mock) and TAB1-2B4 cells (TAB1) were cultured with anti-TCR MAb (TCR) or with PMA (20 ng/ml) and ionomycin (Iono; 1 μM) for 24 h. The levels of IL-2 in the supernatants were determined by an ELISA. Error bars indicate standard deviations. (F) Jurkat cells were transiently transfected with pGL2B luciferase reporter constructs driven by the mouse IL-10 promoter (−1536 to +64), with vector pCMVF, which contained either no insert (Mock) or human TAB1 <t>cDNA</t> (TAB1), and with internal control phRL-TK. Cells were cultured with PMA (100 ng/ml) and ionomycin (2 μM) for 18 h, and luciferase activity was measured.