Figure 3.

The level of expression of a stably integrated gene correlates with the number and strength of the activation domains bound to its promoter. (A) The indicated DNA-binding domain and activation domain fusion proteins (see Materials and Methods for abbreviations used) were transfected into HT1080B cells that carry a stably integrated SEAP reporter gene placed under the control of five GAL4-binding sites. In all cases, SEAP expression values are plotted for cultures receiving 100 ng of activation domain expression plasmid, which gives peak expression values in transiently transfected cells and slightly below peak levels in the stably transfected cell line. The background SEAP activity (24 SEAP units) was subtracted from each value before plotting. In this experiment, expressing the GF1 + RS combination of fusion proteins produced four SEAP units above background levels in the presence of rapamycin. (B) The DNA-binding domain and activation domain expression plasmids were transfected into HT1080B cells. In all cases, mean values of SEAP activity secreted into the medium after the addition of 10 nM rapamycin are shown (±SD). Western blot analysis of the total cell lysates with anti-hemagglutinin antibody allowed an assessment of transcription factor component levels in cells.