Transcript Daryl Khaw Dr. Virginia Weis Department of Zoology

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)