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Searching for TRAF6 in Aiptasia pallida

Searching for TRAF6 in Aiptasia pallida. Daryl Khaw Dr. Virginia Weis Department of Zoology. Purpose of Research in the Weis Lab. http://placestovisitbeforeidie.tumblr.com/. http://www.solcomhouse.com/coralreef.htm. Aiptasia pallida. Clonal reproduction Easily cultured in labs

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Searching for TRAF6 in Aiptasia pallida

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  1. Searching for TRAF6 in Aiptasiapallida Daryl Khaw Dr. Virginia Weis Department of Zoology

  2. Purpose of Research in the Weis Lab http://placestovisitbeforeidie.tumblr.com/ http://www.solcomhouse.com/coralreef.htm

  3. Aiptasiapallida • Clonal reproduction • Easily cultured in labs • Can be bleached and kept aposymbiotic Symbiotic Aposymbiotic

  4. Symbiotic Relationship nutrients alga nutrients symbiosome Algae within gastrodermal cells host cell Nutritional exchange Epidermis Gastric cavity

  5. Innate Immune Response TGFβ Stimulation Suppression RII TLR SMADs NFKB Symbiont Immune Response Host Cell

  6. Toll-Like Receptor Pathway Signal TLR MyD88 TRAF6 TRAF6 IKB NFKB Inflammation Host Cell

  7. Question: Is the TRAF6 protein present in Aiptasiaand, if it is, when is it activated?

  8. Prediction The TRAF6 protein is both present and activated in Aiptasia, but with a greater concentration in immune-stimulated aposymbioticAiptasia TGFβ Suppression Stimulation RII TLR SMADs TRAF6 Symbiont Immune Response Host Cell

  9. Immune-Stimulating Condition: Lipopolysaccharide (LPS) Experimental Group Control Group Symbiotic and aposymbioticAiptasia plated in 24 well plate (3 days FSW) Symbiotic and aposymbioticAiptasia plated in 24 well plate (4 days FSW) Protein Extraction Infected with LPS (12 hrs)

  10. Methods Protein Extraction Step 1: Immunoprecipitation - Isolate and concentrate the TRAF6 protein Step 2: Western Blot Analysis - Detect the presence and size of the TRAF6 protein Immunoprecipitation Result Western Blot

  11. Immunoprecipitation Centrifugation of solution and removal of supernatant Beads bind to protein-antibody complex Protein Extract Add TRAF6 Antibody Antibody binds to TRAF6 protein Add protein G beads

  12. Western Blot Immunoprecipitated Protein SDS polyacrylamide gel electrophoresis Nitrocellulose membrane Run TRAF6 on a gel Transfer to nitrocellulose membrane Nitrocellulose membrane Result Primary Antibody Secondary Antibody Film Exposure Blocking AntiTRAF6

  13. Results AntiTRAF6 TRAF6 Band 60 kDa Heavy Chain Positive Control (Hela cells) Apo LPS Apo Sym LPS Sym

  14. Closer look at the TRAF6 Band 60 kDa Positive Control (Hela cells) Apo LPS Apo Sym LPS Sym

  15. Difficulties and Future Work • Aiptasia are 70% water and contain very little protein • This is an ongoing experiment that will continue during the academic school year: 1. Reproduce the same results using more biological replicates and, if successful, 2. Search for other proteins related to the TLR pathway, such as ubiquitin.

  16. Conclusion TGFβ Based on the information so far: • The TRAF6 protein is present and activated in Aiptasia • The highest concentration of the TRAF6 protein is in immune-stimulated aposymbioticAiptasia Suppression Stimulation RII TLR SMADs TRAF6 Symbiont Immune Response Host Cell

  17. Acknowledgements Thanks to: • Howard Hughes Medical Institute • Cripps Scholarship Fund • Dr. Kevin Ahern The Weis Lab • Dr. Virginia Weis • Angela Poole • Christy Schnitzler • Emilie Fleur • JessiKershner • Olivier Detournay

  18. Results AntiTRAF6 IgG Negative Control TRAF6 Band No TRAF6 Band 60 kDa Heavy Chain Heavy Chain Positive Control (Hela cells) Apo LPS Apo Sym LPS Sym Ladder Apo LPS Apo Sym LPS Sym

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