FIG. 5.

Inhibition of MEKK1-induced transcription by MEK inhibitors. (A) Triplicate cultures of HeLa cells were transiently transfected with a PRE-luciferase reporter construct and PR-B and either pCMV5 control vector or MEKK1. Cells were pretreated for 30 min without or with MEK inhibitors specific for p42 and p44 MAPKs (PD98059; 50 μM) or p38 MAPK (SB203580; 20 μM) and then treated without (black bars) or with (gray bars) R5020 (10 nM) for 12 h. Luciferase activity in cell lysates was determined as described above (see Materials and Methods). MAPK activities were measured in duplicate samples from the same experimental set with phospho-specific MAPK antibodies that recognize only activated p42 and p44 MAPKs or activated p38 MAPK. (B) Phosphorylation of PR Ser294 in the presence of MEKK1 and the p38 MAPK inhibitor (SB580). Duplicate cultures of HeLa cells were transfected with MEKK1 and treated with either dimethyl sulfoxide (vehicle) or the p38 MAPK inhibitor (SB203580; 20 μM) for 12 h. Phospho-Ser294 PR levels in cell lysates were measured with monoclonal antibodies specific to PR phospho-Ser294. Total PR levels in the same lysates were measured (bottom).