FIG. 1.
Dexamethasone-dependent inhibition of p38 is mediated by a stress-activated protein kinase (SAPK)-specific phosphatase(s). (A) HeLa-TO cells were incubated (+) for 2 h with 100 nM dexamethasone (Dex), and the cells were treated (+) with UV C and sodium orthovanadate was added to the medium at the indicated concentrations. After 30 min, total cell lysates were prepared and analyzed by Western blotting with anti-phospho-p38 antibody or total p38 antiserum. (B) HeLa-TO cells were transiently transfected with 500 ng of either pSR-HA-p38 or pSR-HA-p38N316. After 24 h, cells were incubated for 2 h with dexamethasone at the indicated concentrations and then treated with UV C (+) or left untreated (−). After 30 min, total cell lysates were prepared and analyzed by Western blotting with anti-phospho-p38 antibody or HA antibody as a control for transfection. (C) HeLa cells were transiently transfected with 500 ng of pSR-HA-p38 and cotransfected with 20 ng (lane 3), 50 ng (lane 4), 75 ng (lane 5), or 150 ng (lane 6) of pFlagCMV-MKP-1 or 20 ng (lanes 8 and 12), 50 ng (lanes 9 and 13), 75 ng (lanes 10 and 14), or 150 ng (lanes 11 and 15) of pFlagCMV-MKP-1C258S. The empty expression vector pFlagCMV was added as appropriate to bring the total quantity of DNA up to 1 μg. After 24 h, cells were treated as indicated with vehicle (methanol) or 100 nM dexamethasone. After a further 2 h, cells were left untreated or stimulated with UV C, and lysates were prepared 30 min later and subjected to Western blotting using anti-phospho-p38 or anti-HA epitope antibodies. (D) HeLa cells were incubated for 2 h with 100 nM dexamethasone and then stimulated with 10 ng of epidermal growth factor (EGF) per ml for 10 min. Cells were harvested, and total cell lysates were analyzed by Western blotting with anti-phospho-ERK or total ERK antibodies.