TGF-β responsive
co-modulators, Smad2 and 4, increase the activities of both GAL4–MEF2A
and GAL4–MEF2C. (A and B)
C2C12 cells were co-transfected with pCMV5B–TβRI(T204D),
pCMV5B–Smad2, pCMV5B–Smad4, 5×GAL4-luc,
GAL4(DBD), GAL4–MEF2A, GAL4–MEF2C and pSV-βgal as indicated and treated with (+) or without (–) 2 ng/ml of TGF-β in
1% FBS. The transcriptional activation domains of both
MEF2 isoforms are fused to the DNA binding domain (DBD) of GAL4
to form GAL4–MEF2A (91–507 amino acids) and GAL4–MEF2C
(87–442 amino acids). GAL4(DBD) contains the GAL4 DBD only.
The reporter plasmid, 5×GAL4-luc, contains
five copies of the GAL4 binding site linked to the adenovirus EIB
promoter and the firefly luciferase gene. After 72 h, β-galactosidase
activities were measured and used to normalise luciferase activity
values. Two or more sets of assays were performed in both COS and
C2C12 cells with comparable results. Each data point is a mean of
triplicate samples from single experiments and the error bars represent
the standard error of the mean (SEM).