Pap1 pma ta luc

HeLa or A375 cells were seeded in 24-well plates (40 000 cells/well) and incubated for 18 h to achieve ~60–70% confluence. Cells were transfected with AP-1 [pAP1(PMA)-TA-Luc; Clontech] or the serum-response element (SRE; pGL4.33-SRE; Promega) luciferase reporter plasmids (250 ng/well) using Lipofectamine™ (Invitrogen). After 16 h, cells were pre-treated with 1–100 μM compounds as indicated for 20 min, followed by stimulation with 25 ng/ml EGF or 200 nM PMA for 4.5 h. In some experiments, cells were transfected with the pAP1(PMA)-TA-Luc promoter and a constitutively active MEK1 mutant cDNA were treated with various amounts of compounds during the last 4 h of incubation. The luciferase activity in the cell extracts was determined with a Dual Luciferase Assay System (Promega) according to the manufacturer’s instructions. Human embryonic kidney (HEK) 293 cells (20 000 cells/well) were plated in white walled 96-well plates (Nunclon Delta Surface; Thermo Scientific) and then transfected with the luciferase reporter plasmids indicated above. Cells were treated with EGF or PMA for 5 h in the presence or absence of compounds (1–100 μM) and luminescence was measured using Bright-Glo Substrate (Promega). Luciferase activities were monitored with a Lumat LB 9507 luminometer (Berthold Technology) and data were normalized to the amount of protein in each sample.