Fig. 1.
Fig. 1. Importance of tyrosine 693 and serine 721 phosphorylation for IL-12-induced STAT4 activation. / (A), IL-12 induces STAT4 serine phosphorylation. Lymphocytes purified from mouse spleens were activated with ConA for 48 hours. After serum starvation, cells were left unstimulated (−) or were restimulated with IL-12 for the times indicated. Lysates were immunoprecipitated (IP) with anti-STAT4 Ab, resolved by SDS/polyacrylamide gel electrophoresis (SDS/PAGE), transferred to membrane and blotted (IB) with antiphosphoserine 727 STAT3 Ab that also detects serine phosphorylated STAT4 (upper panel). The blot was stripped and reprobed with anti-STAT4, demonstrating equal levels of proteins (lower panel). (B), Tyrosine 693 and serine 721 are important sites of phosphorylation in STAT4. 293T cells were transfected with 0.5 μg pEFBOS-IL-12Rβ1 and 1.5 μg pEFBOS-IL-12Rβ2 alone (lane 1) or together with 0.5 μg wild-type STAT4 (lanes 2-4), STAT4 Y693F (lanes 5-7) or STAT4 S721A (lanes 8-10). Cells were left unstimulated (−) or were stimulated with IL-12 for the times indicated. Lysates were immunoprecipitated with anti-STAT4 Ab, resolved by SDS/PAGE and blotted sequentially with antiphosphotyrosine Ab (upper panel) and anti-STAT4 (lower panel). (C), Tyrosine 693 and serine 721 are important for IL-12–mediated, STAT4-dependent transcriptional activity. Jurkat T cells were transiently transfected with the p3xGAS-luc and the pCMV-β-galactosidase reporters and expression vectors encoding the human IL-12R subunits. Where indicated, cells were cotransfected with control vector (EV), wild-type STAT4, or mutated STAT4 constructs. Transfected cells were left untreated or were stimulated with IL-12 before lysis. Luciferase and β-galactosidase activities were measured as described in “Materials and methods.” The data represent luciferase activity normalized by the β-galactosidase activity present in each sample, expressed as fold induction relative to the control, and are the averages ± SD of triplicate samples from a typical experiment. Unstimulated, ■; +IL-12, ▪.

Importance of tyrosine 693 and serine 721 phosphorylation for IL-12-induced STAT4 activation.

(A), IL-12 induces STAT4 serine phosphorylation. Lymphocytes purified from mouse spleens were activated with ConA for 48 hours. After serum starvation, cells were left unstimulated (−) or were restimulated with IL-12 for the times indicated. Lysates were immunoprecipitated (IP) with anti-STAT4 Ab, resolved by SDS/polyacrylamide gel electrophoresis (SDS/PAGE), transferred to membrane and blotted (IB) with antiphosphoserine 727 STAT3 Ab that also detects serine phosphorylated STAT4 (upper panel). The blot was stripped and reprobed with anti-STAT4, demonstrating equal levels of proteins (lower panel). (B), Tyrosine 693 and serine 721 are important sites of phosphorylation in STAT4. 293T cells were transfected with 0.5 μg pEFBOS-IL-12Rβ1 and 1.5 μg pEFBOS-IL-12Rβ2 alone (lane 1) or together with 0.5 μg wild-type STAT4 (lanes 2-4), STAT4 Y693F (lanes 5-7) or STAT4 S721A (lanes 8-10). Cells were left unstimulated (−) or were stimulated with IL-12 for the times indicated. Lysates were immunoprecipitated with anti-STAT4 Ab, resolved by SDS/PAGE and blotted sequentially with antiphosphotyrosine Ab (upper panel) and anti-STAT4 (lower panel). (C), Tyrosine 693 and serine 721 are important for IL-12–mediated, STAT4-dependent transcriptional activity. Jurkat T cells were transiently transfected with the p3xGAS-luc and the pCMV-β-galactosidase reporters and expression vectors encoding the human IL-12R subunits. Where indicated, cells were cotransfected with control vector (EV), wild-type STAT4, or mutated STAT4 constructs. Transfected cells were left untreated or were stimulated with IL-12 before lysis. Luciferase and β-galactosidase activities were measured as described in “Materials and methods.” The data represent luciferase activity normalized by the β-galactosidase activity present in each sample, expressed as fold induction relative to the control, and are the averages ± SD of triplicate samples from a typical experiment. Unstimulated, ■; +IL-12, ▪.

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