Type of Submission

Poster

Keywords

Tetrahymena, netrin, Western

Abstract

The netrin family of proteins has homeostatic roles in vertebrate development and angiogenesis, and pathophysiological roles in the progression and metastasis of cancer. We have previously characterized a netrin-1-like protein in Tetrahymena thermophila, and have shown that vertebrate netrin-1, netrin-3, and netrin-4 all serve as chemorepellents in this organism. We are currently using Western blotting and immunofluorescence to further characterize the netrin-like proteins in Tetrahymena. Western blotting with our anti-netrin-4 antibody shows a band that is clearly visible in whole cell extract, but shows little reactivity with secreted protein, indicating that most of our netrin-4-like protein remains within the cell. Western blotting of whole cell extract with anti-netrin-1, netrin-3, and netrin-4 antibodies shows a clear band measuring 50 kD that stains with all three antibodies. Some lower molecular weight bands are also evident in all three blots, possibly due to proteolytic activity. Immunolocalization with an anti-netrin-4 antibody shows some colocalization with netrin-1, netrin-3, and ER Tracker™. Our anti-netrin-4 antibody localizes to the oral groove, basal bodies, and nuclei of cells, indicating a possible structural role for the netrin-4-like protein in this organism. Further research will involve determining the primary amino acid sequence of the 50 kD protein and comparing it with the Tetrahymena thermophila proteome database to help ascertain the physiological role of this protein.

Campus Venue

Stevens Student Center Lobby

Location

Cedarville, OH

Start Date

4-3-2019 11:00 AM

End Date

4-3-2019 2:00 PM

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

Included in

Biochemistry Commons

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Apr 3rd, 11:00 AM Apr 3rd, 2:00 PM

Characterization of a Netrin-4 Like Protein in Tetrahymena thermophila

Cedarville, OH

The netrin family of proteins has homeostatic roles in vertebrate development and angiogenesis, and pathophysiological roles in the progression and metastasis of cancer. We have previously characterized a netrin-1-like protein in Tetrahymena thermophila, and have shown that vertebrate netrin-1, netrin-3, and netrin-4 all serve as chemorepellents in this organism. We are currently using Western blotting and immunofluorescence to further characterize the netrin-like proteins in Tetrahymena. Western blotting with our anti-netrin-4 antibody shows a band that is clearly visible in whole cell extract, but shows little reactivity with secreted protein, indicating that most of our netrin-4-like protein remains within the cell. Western blotting of whole cell extract with anti-netrin-1, netrin-3, and netrin-4 antibodies shows a clear band measuring 50 kD that stains with all three antibodies. Some lower molecular weight bands are also evident in all three blots, possibly due to proteolytic activity. Immunolocalization with an anti-netrin-4 antibody shows some colocalization with netrin-1, netrin-3, and ER Tracker™. Our anti-netrin-4 antibody localizes to the oral groove, basal bodies, and nuclei of cells, indicating a possible structural role for the netrin-4-like protein in this organism. Further research will involve determining the primary amino acid sequence of the 50 kD protein and comparing it with the Tetrahymena thermophila proteome database to help ascertain the physiological role of this protein.

 

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