1. UCHC High School Reasearch
Apprenticeship Program
Caroline Kuzoian and Anurag Ojha

2. • Cell Migration is found in:
• Embryonic Development
• Wound healing
• Cancer spread (metastasis)
• PE (Parietal Endoderm) cells
• First migratory embryonic cells
• Derived by F9 cells
• Interaction helps cells migrate properly to form
yolk sac surrounding embryo

3. • To determine the presence and function of the Nectin-
Afadin complex

4. F9 Primitive Endoderm PE/VE

5. • Nectin – trans-membrane protein, extracellular
component of the Nectin-Afadin complex
• Afadin – intracellular component of Nectin-Afadin
complex, binding to the cytoskeleton
• Partitioning Defective 3 (Par-3) – binds to nectin and
recruits afadin to create the Nectin-Afadin complex
• Actin – a major component of the cytoskeleton and is
involved in cell-to-cell interactions

6. • Par-3 binds to nectin, nectin binds to afadin, which binds
to actin
• This causes actin to reorganize and recruit inactive E-Cadherins to
the binding site
• E-Cadherins create a strong bond between cells in the adherens
junction

7. • Small interfering
• A technique used to decrease the translation of a
targeted protein
• Results in double stranded mRNA which cannot be translated
completely

8. • Primary
• Secondary
• Tags
• Horseradish Peroxidase – Western blotting
• Alexa fluor – Immunofluorescence
• Beads coated in A/G protein – Immunoprecipitation

9. • A process to determine presence of specific proteins
within a cell lysate
• Used to compare presence of same proteins within different cell
types
• Proteins are identified by their molecular weight

10. • Lyse=burst
• Isolate
proteins
Cell
Lysis
• Find unknown protein
concentration
Bradford
Assay
• Run current through
gel
• Separate proteins by
size
Loading
Gel
• Run current
• Proteins pulled onto
membrane
Semi-
Dry
Blotting
• Incubate overnight
• Wash
Primary
Antibody
• Incubate one hour
• Wash
Secondar
y
Antibody
• Substrate produces
color
OPTI-
4CN
Stain
Protein
Protein

11. Protein Molecular
Weight
(kDa)
Nectin 87
Afadin 200
Par-3 180, 150,
100
Actin 42
Probed by afadin
antibody 7/17
Probed by actin
antibody 7/17
Control Lipofectamine siScramble siAfadin
siPar-3
Control
200
kD
a
42
kD
a
Control Lipofectamine siScramble siAfadin
siPar-3
Control

12. • Biotinylation – process in which biotin, a coenzyme,
attaches to all the proteins present on the cell surface
• Immunoprecipitation - Primary and secondary antibodies
bind to target protein and pull down the protein of
interest with the help of a bead coated in an A/G protein

15. • More immunofluorescence staining of siRNA treated cells
to determine functional changes of the Nectin-Afadin
complex
• More western blotting to determine changes of levels in
par3 and nectin after siRNA treatment

16. We would like to acknowledge the support of:
• The Department of Health Career Opportunities
• Aetna Health Professions Partnership Initiative.
• Theo Szmurlo
• David Artus
• Alexander “The Great” Pokorski
• Nathaniel Aponte
• Dr. Kathy Martin PhD.
• Dr. Jim Mulrooney PhD.