Download - Searching for TRAF6 in Aiptasia pallida
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