Fig. 3.
Fig. 3. IL-12 activates p38, but not ERKs or JNKs in non-lymphoid cells; serine 721 is important for IL-12-induced, STAT4-mediated transactivation in a heterologous system. / (A), IL-12 does not activate ERK2. NIH3T3 were transiently transfected with expression vectors encoding the human IL-12R subunits and pCDNA3-HA-ERK2. Cells were left unstimulated (lane 1) or were stimulated with serum (lane 2) or IL-12 for the times indicated (lanes 3 and 4). Lysates were immunoprecipitated with anti-HA Ab and a kinase reaction was performed on the immunoprecipitates using MBP as a substrate (upper panel). The filter was probed for ERK2 showing equal loading of the proteins (lower panel). (B), IL-12 does not activate JNK1. NIH3T3 were transiently transfected with expression vectors encoding the human IL-12R subunits and pCDNA3-HA-JNK1. Cells were left unstimulated (lane 1) or were stimulated with 10 μg/mL anisomycin (lane 2) or with IL-12 for the times indicated (lanes 3 and 4). Lysates were immunoprecipitated with anti-HA Ab. A kinase reaction was performed on the immunoprecipitates using GST-ATF2 as a substrate (upper panel). The filter was probed for JNK1 showing equal loading of the proteins (lower panel). (C), Activation of p38 by IL-12. NIH3T3 were transiently transfected with expression vectors encoding the human IL-12R subunits and pCEFL-HA-p38α. Cells were left unstimulated (lane 1) or were stimulated with 10 μg/mL anisomycin (lane 2) or with IL-12 for the times indicated (lanes 3-6). Lysates were immunoprecipitated with anti-HA Ab. A kinase reaction was performed on the immunoprecipitates using MBP as a substrate (upper panel). The filter was probed for p38 showing equal loading of the proteins (lower panel). (D), Importance of tyrosine 693 and serine 721 for IL-12–induced STAT4 transcriptional activity. NIH3T3 cells were transiently transfected with the p3xGAS-luc and the pCMV-β-galactosidase reporters together with expression vectors encoding the human IL-12R subunits. Where indicated, cells were cotransfected with control vector (EV), wild-type STAT4, or the mutated STAT4 constructs. The experiment was performed as described in Figure 1C. Unstimulated, ■; +IL-12, ▪. In the lower panel, 100 μg of the cell lysates, analyzed in the luciferase assay, were resolved by SDS/PAGE, transferred to membrane, and blotted with anti-STAT4 Ab.

IL-12 activates p38, but not ERKs or JNKs in non-lymphoid cells; serine 721 is important for IL-12-induced, STAT4-mediated transactivation in a heterologous system.

(A), IL-12 does not activate ERK2. NIH3T3 were transiently transfected with expression vectors encoding the human IL-12R subunits and pCDNA3-HA-ERK2. Cells were left unstimulated (lane 1) or were stimulated with serum (lane 2) or IL-12 for the times indicated (lanes 3 and 4). Lysates were immunoprecipitated with anti-HA Ab and a kinase reaction was performed on the immunoprecipitates using MBP as a substrate (upper panel). The filter was probed for ERK2 showing equal loading of the proteins (lower panel). (B), IL-12 does not activate JNK1. NIH3T3 were transiently transfected with expression vectors encoding the human IL-12R subunits and pCDNA3-HA-JNK1. Cells were left unstimulated (lane 1) or were stimulated with 10 μg/mL anisomycin (lane 2) or with IL-12 for the times indicated (lanes 3 and 4). Lysates were immunoprecipitated with anti-HA Ab. A kinase reaction was performed on the immunoprecipitates using GST-ATF2 as a substrate (upper panel). The filter was probed for JNK1 showing equal loading of the proteins (lower panel). (C), Activation of p38 by IL-12. NIH3T3 were transiently transfected with expression vectors encoding the human IL-12R subunits and pCEFL-HA-p38α. Cells were left unstimulated (lane 1) or were stimulated with 10 μg/mL anisomycin (lane 2) or with IL-12 for the times indicated (lanes 3-6). Lysates were immunoprecipitated with anti-HA Ab. A kinase reaction was performed on the immunoprecipitates using MBP as a substrate (upper panel). The filter was probed for p38 showing equal loading of the proteins (lower panel). (D), Importance of tyrosine 693 and serine 721 for IL-12–induced STAT4 transcriptional activity. NIH3T3 cells were transiently transfected with the p3xGAS-luc and the pCMV-β-galactosidase reporters together with expression vectors encoding the human IL-12R subunits. Where indicated, cells were cotransfected with control vector (EV), wild-type STAT4, or the mutated STAT4 constructs. The experiment was performed as described in Figure 1C. Unstimulated, ■; +IL-12, ▪. In the lower panel, 100 μg of the cell lysates, analyzed in the luciferase assay, were resolved by SDS/PAGE, transferred to membrane, and blotted with anti-STAT4 Ab.

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