We next carried out in vitro kinase assays using GST-ATF2 as a substrate to further confirm that DUSP26 regulates p38 activity and this GDC-0941 inhibitory effect is suppressed by NSC-87877. DUSP26 decreased the kinase activity of p38 and this inhibitory effect was blocked in the presence of NSC-87877 ( Fig. 3C, lanes 1–4). Since DUSP26 has no phosphatase activity on phospho-ATF2 ( Fig. 3C, lanes 5 and 6), these data suggest that DUSP26 specifically dephosphorylates and inhibits p38 and NSC-87877 inhibits the action of DUSP26 on p38.
Fig. 4.
NSC-87877 inhibits the action of DUSP26 on p38 in vivo. (A) Transfected HEK 293 cells were pretreated with various concentrations of NSC-87877 (0–100 μM) for 3 h and then stimulated with H2O2 (1 mM, 30 min). Cell lysates were analyzed by immunoblotting with appropriate antibodies as described. (B) After transfection, HEK 293 cells were pretreated with various concentrations of NSC-87877 (0–100 μM) for 3 h and then stimulated with H2O2 (1 mM, 30 min). Immunoprecipitation and immunoblotting analyses were processed as in Materials and methods. The immune complex kinase assay was carried out as described under Materials and methods. The relative p38 kinase activity is shown.
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