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 Can be bleached and kept aposymbiotic Symbiotic Aposymbiotic Symbiotic Relationship nutrients alga nutrients symbiosome Algae within gastrodermal cells host cell Nutritional exchange Epidermis Gastric cavity Innate Immune Response TGFβ Stimulation Suppression RII TLR SMADs NFKB Host Cell Immune Response Symbiont Toll-Like Receptor Pathway Signal TLR MyD88 TRAF6 IKB NFKB Inflammation Host Cell Question: Is the TRAF6 protein present in Aiptasia and, if it is, when is it activated? Prediction The TRAF6 protein is both present and activated in Aiptasia, but with a greater concentration in immunestimulated aposymbiotic Aiptasia TGFβ Suppression Stimulation RII TLR SMADs TRAF6 Immune Response Host Cell Symbiont Immune-Stimulating Condition: Lipopolysaccharide (LPS) Experimental Group Symbiotic and aposymbiotic Aiptasia plated in 24 well plate (3 days FSW) Control Group Symbiotic and aposymbiotic Aiptasia plated in 24 well plate (4 days FSW) Protein Extraction Infected with LPS (12 hrs) Methods Protein Extraction Immunoprecipitation Western Blot Step 1: Immunoprecipitation - Isolate and concentrate the TRAF6 protein Step 2: Western Blot Analysis - Detect the presence and size of the TRAF6 protein Result Immunoprecipitation Protein Extract Add TRAF6 Antibody Antibody binds to TRAF6 protein Add protein G beads Beads bind to protein-antibody complex Centrifugation of solution and removal of supernatant Western Blot Immunoprecipitated Protein SDS polyacrylamide gel electrophoresis Run TRAF6 on a gel Nitrocellulose membrane Transfer to nitrocellulose membrane Nitrocellulose membrane Result Blocking Primary Antibody AntiTRAF6 Secondary Film Antibody Exposure Results AntiTRAF6 TRAF6 Band 60 kDa Heavy Chain Positive Control (Hela cells) Apo LPS Apo Sym LPS Sym Closer look at the TRAF6 Band 60 kDa Positive Control (Hela cells) Apo LPS Apo Sym LPS Sym 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. Conclusion TGFβ Based on the information so far: Suppression 1. 2. The TRAF6 protein is present and activated in Aiptasia The highest concentration of the TRAF6 protein is in immune-stimulated aposymbiotic Aiptasia Stimulation RII TLR SMADs TRAF6 Immune Response Host Cell Symbiont 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 Jessi Kershner Olivier Detournay Results AntiTRAF6 IgG Negative Control TRAF6 Band No TRAF6 Band 60 kDa Heavy Chain Heavy Chain LPS Sym Sym LPS Apo Apo Ladder LPS Sym Sym LPS Apo Apo Positive Control (Hela cells)