Fig. 1

Design of and functional studies with the expression constructs MA4, MA4m and A4M. A Design of the A4M, MA4 and MA4m transgenes. Amino acids coordinates of fused portions and the pSer domain are indicated. The exchanged pSer domain contained the binding sites for the Mediator complex, SL1 and TBP. The helical wheel presentation of a portion of this sequence is shown below. This allows to visualize the differences between both sequences and consequences of missing or additional amino acids. B RT-PCR analyses of all inducible transgenes. These experiments validated the correct expression of all stably transfected transgenes. Sizes of each amplimer are given to the left. A GAPDH primer set was used to validate that equal amounts of cellular RNA were used in all experiments. C Cell viability and target gene validation. Cell viability was tested in independent experiments (n = 3). A single downstream target genes, HOXA9, was tested to validate the functionality of both the MA4 and MA4m fusion gene constructs. D Co-immunoprecipitation validated the binding of SL1 to the human pSer domain, while the murinized version binds to a eightfold lesser extent. Below: analyses demonstrating equal transcription of both MTM-(m)pSer domains (RT-PCR), and equal expression by Western blot analysis (WBA) E MACE-Seq experiment revealed the synergism between MA4 and A4M. MA4m has lost this ability