Transcriptional activation of BRE-dependent transcription by EHZF. P19 cells or 293 were transfected with a β-galactosidase–expression plasmid as an internal control, in addition to the luciferase reporter plasmids and plasmids containing the cDNA for the transcription factors indicated. After 18 hours the medium was changed, and at 36 hours cells were stimulated with BMP2/4 or TGF-β. Extracts were prepared and assayed for β-galactosidase 48 hours after transfection. Based on its levels, normalized amounts of extracts were assayed for luciferase activity. The results shown are the average of triplicates from at least 2 independent experiments. Error bars indicate standard deviations. (A) P19 cells were cotransfected with pBRE-Luc and either the control vector p3xFlagCMV7.1, pFlagCS2-OAZ(KIAA0760-L), or p3xFlagCMV7.1-EHZF for 36 hours and were then incubated with either 50 ng/mL BMP2, 25 ng/mL BMP4, or 5ng/mL TGF-β for 16 hours in the presence of reduced serum concentration. (B,C) 293 and P19 cells were cotransfected with either the control vector alone, pFlagCS2-OAZ(KIAA0760-L) 500 ng, p3xFlagCMV7.1-EHZF 500 ng, or pFlagCS2-OAZ(KIAA0760-L) 500 ng with the addition of either 1. (500 ng), 2. (1 μg), 3. (2 μg), 4. (4 μg) of p3xFlagCMV7.1-EHZF, for 36 hours and when indicated exposed to 25 ng/mL BMP4 for 16 hours in 0.5% serum. Cell extracts were normalized for β-galactosidase activity and assayed for luciferase activity. The 293 cells were transfected with either the vector or EHZF in the presence of (D) pCRE-Luc (with or without forskolin, 10^–5M); (E) pARE-Luc (with or without cotransfected pEF-flag-Mixer), (F) pDE-Luc (with or without cotransfected pEF-Flag-Fast-1) for 36 hours and then when indicated treated with 5 ng/mL TGF-β in 0.5% serum for 16 hours.