Fig. 2.
Fig. 2. IL-12 activates p38 but not ERKs or JNKs in T and NK3.3 cells. / (A), IL-2 but not IL-12 induces Shc phosphorylation. T cells were left unstimulated (lane 1) or were stimulated for 15 minutes with 10 ng/mL IL-12 (lane 2) or 2000 IU/mL IL-2 (lane 3). Lysates were immunoprecipitated with anti-Shc Ab, resolved by SDS/PAGE, transferred to membrane, and blotted with antiphosphotyrosine Ab (upper panel). The blot was stripped and reprobed with anti-Shc, demonstrating equal levels of proteins (lower panel). (B), IL-2 but not IL-12 induces Shc/Grb2 association. T cells were left unstimulated (lane 1) or were stimulated for 15 minutes with IL-2 (lane 2) or IL-12 (lane 3). Lysates were immunoprecipitated with anti-Shc Ab, resolved by SDS/PAGE, and blotted sequentially with anti-Grb2 (upper panel) and anti-Shc Ab (lower panel). (C), IL-2 but not IL-12 activates ERK2. T cells were left unstimulated (lane 1) or were stimulated for 15 minutes with IL-2 (lane 2) or IL-12 (lane 3). Lysates were immunoprecipitated with anti-ERK2 Ab and a kinase reaction was performed on the immunoprecipitates using MBP as a substrate (upper panel). Parallel samples were immunoprecipitated with anti-ERK2 and blotted with antiphosphotyrosine (middle panel) and anti-ERK2 Ab (lower panel). (D), A MEK inhibitor does not affect STAT4 serine phosphorylation. T cells were left unstimulated (lanes 1 and 5) or stimulated with IL-12 (lanes 2-4 and 6-8) for different periods. Cells in lanes 5 to 8 were pretreated with 100 μmol/L PD98059 for 30 minutes. Cell lysates were immunoprecipitated with anti-STAT4 Ab, resolved by SDS/PAGE, and blotted with anti-STAT4. (E), IL-12 does not activate JNK1. NK3.3 cells were left unstimulated (lane 1) or were stimulated with 10 μg/mL anisomycin (lane 2), IL-2 (lane 3), or IL-12 (lane 4). Lysates were immunoprecipitated with anti-JNK1 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). (F), Activation of p38 by IL-12. NK3.3 cells were left unstimulated (lanes 1 and 6) or were stimulated with IL-12 for different periods (lanes 2-5) or with 10 μg/mL anisomycin for 15 minutes (lane 7). Lysates were immunoprecipitated with anti-p38 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).

IL-12 activates p38 but not ERKs or JNKs in T and NK3.3 cells.

(A), IL-2 but not IL-12 induces Shc phosphorylation. T cells were left unstimulated (lane 1) or were stimulated for 15 minutes with 10 ng/mL IL-12 (lane 2) or 2000 IU/mL IL-2 (lane 3). Lysates were immunoprecipitated with anti-Shc Ab, resolved by SDS/PAGE, transferred to membrane, and blotted with antiphosphotyrosine Ab (upper panel). The blot was stripped and reprobed with anti-Shc, demonstrating equal levels of proteins (lower panel). (B), IL-2 but not IL-12 induces Shc/Grb2 association. T cells were left unstimulated (lane 1) or were stimulated for 15 minutes with IL-2 (lane 2) or IL-12 (lane 3). Lysates were immunoprecipitated with anti-Shc Ab, resolved by SDS/PAGE, and blotted sequentially with anti-Grb2 (upper panel) and anti-Shc Ab (lower panel). (C), IL-2 but not IL-12 activates ERK2. T cells were left unstimulated (lane 1) or were stimulated for 15 minutes with IL-2 (lane 2) or IL-12 (lane 3). Lysates were immunoprecipitated with anti-ERK2 Ab and a kinase reaction was performed on the immunoprecipitates using MBP as a substrate (upper panel). Parallel samples were immunoprecipitated with anti-ERK2 and blotted with antiphosphotyrosine (middle panel) and anti-ERK2 Ab (lower panel). (D), A MEK inhibitor does not affect STAT4 serine phosphorylation. T cells were left unstimulated (lanes 1 and 5) or stimulated with IL-12 (lanes 2-4 and 6-8) for different periods. Cells in lanes 5 to 8 were pretreated with 100 μmol/L PD98059 for 30 minutes. Cell lysates were immunoprecipitated with anti-STAT4 Ab, resolved by SDS/PAGE, and blotted with anti-STAT4. (E), IL-12 does not activate JNK1. NK3.3 cells were left unstimulated (lane 1) or were stimulated with 10 μg/mL anisomycin (lane 2), IL-2 (lane 3), or IL-12 (lane 4). Lysates were immunoprecipitated with anti-JNK1 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). (F), Activation of p38 by IL-12. NK3.3 cells were left unstimulated (lanes 1 and 6) or were stimulated with IL-12 for different periods (lanes 2-5) or with 10 μg/mL anisomycin for 15 minutes (lane 7). Lysates were immunoprecipitated with anti-p38 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).

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