Fig. 1. TTF-1 activation of the Tg and synthetic target promoters is PKA
dependent in A126 cells. A, A126 cells were stably
transfected with pRSV-TTF1 and pRSV-NEO. A pool of 10 clones expressing
TTF1 was analyzed by Western blot analysis, using specific antibodies.
Clones expressing TTF1 were further analyzed by transient transfection
with pTg-CAT, pCRE-CAT, and C-PKA. B, A126 cells were
transiently transfected with pRSV-TTF1, pTg-CAT, and C-PKA expressing
vectors. C, A126 cells were transfected with a synthetic
TTF1 target promoter (pTgTTF1-CAT) as described in the text
with () or without () cotransfection with a C-PKA expression
vector. The total amount of transfected DNA was adjusted to 40 µg/ml
with salmon sperm DNA. A pRSV-LacZ (2.5 µ g/ml) reporter gene was
included to normalize for transfection efficiency. CAT activities were
quantified by ß-scope and normalized to ß-galactosidase activities.
The activity of pRSV-CAT was taken as 100%. Results are expressed as
the percentage of enzymatic activity and represent the means of three
independent experiments; bars, SD. Stimulation by PKA of
TTF1 activity was inhibited by a PKA-specific inhibitor PKI (see
"Materials and Methods").