Post #2: Working with RTH Mutants!

Hi everyone! Since my last post, I have been working on my resistance to thyroid hormone syndrome (RTH) experiments. I expressed my two TR-a1 mutants in a human cell line, and analyzed them under fluorescence microscope. In order to compare the nuclear localization of these mutants to the wild-type receptor, I need to quantify the fluorescence in the nucleus and in the cytoplasm, and obtain a ratio between these values. This value is known as the N/C ratio, and has been used by previous members in the Allison lab to study the intracellular localization patterns of several TR mutants.

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Figure 1. Using fluorescence microscopy, I am able to quantify the concentration of protein inside the nucleus and in the cytoplasm. Then, by obtaining a ratio between these values (the N/C ratio), I can compare the intracellular localization patterns between various TR-a1 variants. The nucleus is stained by a blue-fluorescent DNA-binding stain, while the receptor is tagged with green-fluorescent protein.

Although I still need to complete one more replicate, preliminary results show that both mutants have similar localization patterns as the wild-type TR-a1. I suspect that because the mutations occur in just one of the receptor’s nuclear export signals (NES), and not in the receptor’s nuclear import signals (NIS), they are not enough to cause a significant change in shuttling patterns of TR-a1.

I am currently in the process of performing my final replicate, after which I will be able to draw more conclusions. Stay tuned for my results!

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