TetR is a kind of protein that can find and combined with TRE DNA structure domain. ELK is a kind of protein that can activate downstream gene expression after being regulated by phosphorylation
TetR can recognize and combine with TRE, it is a important part of Tet-off system. Without tetracycline, TetR can combine with TRE. With the presence of tetracycline, the combination of TetR and TRE will be blocked and the Tet-off system will be shut down. ELK is generally dormant, when INSR receive insulin and activate MAPK, ELK will be phophated and activated, then ELK is able activate expression of target gene downstream TRE.
Fig.1 The model diagram TetR-ELK
After TetR binds to TRE, phosphorylation ELK activates downstream reporting gene mCherry expression, and we observe the expression with laser confocal microscope.
We also use ERK antibodies to detect phosphorylation of ERK1/ERK2 in the phosphorylation pathway through Western Blot.
Fig.2 Under Laser confocal microscopy, fluorescence of mCherry expression downstream of Tet-Off system
Fig.3 ERK phosphorylation changes with different insulin treatment
Fig.4 ERK phosphorylation changes with different insulin treatment
Despite the length and the complication of phosphorylation pathway, the phosphorylation pathway of protein is a very short process, it is usually completed within minutes even seconds. The time of cracking the cells and collecting protein must be controlled precisely when detecting
Haifeng Ye, Mingqi Xie, Shuai Xue.Self-adjusting synthetic gene circuit for correcting insulin resistance[J].Nat Biomed Eng.2017 Jan;1(1):0005.
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