Searching for TRAF6 in Aiptasia pallida

Daryl KhawDr. Virginia Weis

Department of Zoology

Purpose of Research in the Weis Lab

http://placestovisitbeforeidie.tumblr.com/ http://www.solcomhouse.com/coralreef.htm

Aiptasia pallidaClonal reproductionEasily cultured in labsCan be bleached and kept aposymbiotic

Symbiotic Aposymbiotic

Symbiotic Relationship

Epidermis

Algae within gastrodermal cells

Gastric cavity

symbiosomehost cell

nutrients

nutrients

alga

Nutritional exchange

Innate Immune Response

TLR

NFKB

RII

TGFβ

SMADs

Immune Response

Stimulation Suppression

Host CellSymbiont

Toll-Like Receptor PathwayTLR

IKB

NFKB

Inflammation

MyD88 TRAF6

TRAF6

Host Cell

Signal

Question:Is the TRAF6 protein present in Aiptasia and, if it is, when is it activated?

PredictionThe TRAF6 protein is

both present and activated in Aiptasia, but with a greater concentration in immune-stimulated aposymbiotic Aiptasia TLR

TRAF6

Immune Response

RII

TGFβ

SMADs

Host Cell

Stimulation

Suppression

Symbiont

Immune-Stimulating Condition: Lipopolysaccharide (LPS)

Experimental Group Control Group

Symbiotic and aposymbiotic Aiptasia plated in 24 well plate (3

days FSW)

Infected with LPS (12 hrs)

Symbiotic and aposymbiotic Aiptasia plated in 24 well plate (4

days FSW)

Protein Extracti

on

Methods

Step 1: Immunoprecipitation - Isolate and concentrate the TRAF6 protein

Step 2: Western Blot Analysis - Detect the presence and size of the TRAF6 protein

Protein Extracti

onImmunoprecipitati

on

Western Blot Result

Immunoprecipitation

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

Protein

Extract

Western Blot

Run TRAF6 on a gel

Transfer to nitrocellulose

membrane

Immunoprecipitated Protein

SDS polyacrylamide gel electrophoresis

Nitrocellulose membrane

Nitrocellulose membrane

BlockingPrimary Antibody

Secondary Antibody

Film Exposure

Result

AntiTRAF6

Results

Positive Control

(Hela cells)Apo LPS Apo Sym LPS Sym

Heavy Chain

60 kDa

TRAF6 Band

AntiTRAF6

Closer look at the TRAF6 Band

Positive Control(Hela cells)

Apo LPS Apo Sym LPS Sym

60 kDa

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

Based on the information so far:

1. The TRAF6 protein is present and activated in Aiptasia

2. The highest concentration of the TRAF6 protein is in immune-stimulated aposymbiotic Aiptasia

TLR

Immune Response

Host Cell

Stimulation

Symbiont

TRAF6

RII

TGFβ

Suppression

SMADs

AcknowledgementsThanks to: Howard Hughes Medical InstituteCripps Scholarship FundDr. Kevin Ahern

The Weis LabDr. Virginia WeisAngela PooleChristy SchnitzlerEmilie FleurJessi KershnerOlivier Detournay

ResultsAntiTRAF6 IgG Negative Control

Positive C

ontrol(H

ela cells)

Apo

LPS A

po

Sym LPS Sym

Ladder A

po

LPS Apo

SymLPS Sym

TRAF6

Band60

kDa

Heavy Chain

Heavy Chain

No TRAF6 Band