performed
yeast
two-hybrid
study
identify
proteins
interact
exon11
brca1
identified
protein
phosphatase
pp1β
isoform
serine
threonine
phosphatase
pp1
used
yeast
two-hybrid
assay
detect
proteins
interact
exon11
brca1
large
exon
encodes
roughly
protein
wished
identify
potentially
important
interacting
proteins
outside
intensely
studied
ring
c-terminal
regions
brca1
performed
yeast
two-hybrid
assay
identify
proteins
interact
exon11
brca1
study
putative
positives
identified
including
pp1β
isoform
serine/threonine
phosphatase
pp1
shown
initial
yeast
two-hybrid
studies
led
examination
interaction
brca1
pp1β
performed
yeast
two-hybrid
study
identify
proteins
interact
exon11
brca1
region
brca1
encoded
exon
known
interact
number
proteins
involved
dna
repair
well
γ-tubulin
several
kinases
including
aurora-a
kinase
chkii
identification
additional
interacting
partners
particularly
ones
modify
activity
brca1
changes
phosphorylation
may
aid
clarifying
function
regulation
yeast
two-hybrid
study
identified
serine/threonine
phosphatase
pp1β
brca1
interacting
protein
important
consequences
both
activity
brca1
regulation
pp1β
activitytwo-hybrid
approach
identify
prefoldin-like
protein
ubiquitously
expressed
transcript
uxt
expressed
predominantly
interacts
specifically
nf-κb
inside
nucleus
rna
interference
knockdown
uxt
leads
impaired
nf-κb
activity
dramatically
attenuates
expression
nf-κb
dependent
genes
interference
also
sensitizes
cells
apoptosis
tumor
necrosis
factor-α
identify
new
components
nf-κb
enhanceosome
performed
systematic
yeast
two-hybrid
screening
cdna
fragment
harboring
rhd
p65
amino
acids
used
bait
several
positive
clones
identified
encode
full-length
uxt
fig.
addition
previously
confirmed
p65-interacting
proteins
e.g.
iκbα
pias3
screened
out
interaction
between
p65
uxt
yeast
two-hybrid
assayyeast
two-hybrid
system
detected
interaction
between
co
cop1
although
interaction
between
cop1
co-related
protein
co-like3
col3
previously
detected
method
datta
et
al
able
confirm
interactionhelp
identify
factors
might
shuttled
from
cytosol
er
get
system
performed
yeast
two-hybrid
y2h
screen
polypeptides
interact
get3
y2h
analysis
reports
weak
interactions
occurring
within
nucleus
assayed
strains
well
suited
identifying
get3
binding
proteins
detect
transient
interactions
independent
presence
get1
get2
used
yeast
expressing
get3
bait
screen
genomic
library
encoding
prey
proteins
james
et
al.
physical
interactions
caused
activation
gal4-driven
his3
reporter
gene
allowing
growth
plates
lacking
histidine
strongest
hit
from
screen
fragment
sed5
amino
acid
terminus
figure
2a
ta
protein
acts
snare
vesicular
traffic
within
golgi
between
golgi
er
hardwick
pelham
get3-sed5
interaction
dependent
presence
c-terminal
tmd
figure
2a
consistent
idea
directed
y2h
approach
detected
physical
interactions
between
get3
several
additional
secretory
pathway
ta
proteins
including
snares
tlg2
sec22
peroxisomal
ta
protein
pex15
interactions
observed
sed5
dependent
presence
c-terminal
tmd
figure
3a
first
y2h
analysis
indicates
get3
bind
multiple
secretory
pathway
ta
proteins
tmd-dependent
manner
yeast
two-hybrid
assay
get3
bait
sed5197
strongest
hit
from
y2h
screen
prey
presence
absence
tmd
growth
medium
lacking
histidine
indicative
physical
interaction
y2h
assay
showing
get3
bait
various
ta
proteins
presence
absence
tmds
prey
growth
medium
lacking
histidine
indicative
physical
interactionexplore
potential
targets
p30
during
infection
used
yeast
two-hybrid
system
screen
porcine
macrophage
natural
viral
host
cell
cdna
library
cellular
proteins
may
interact
p30
identified
heterogeneous
nuclear
ribonucleoprotein
hnrnp-k
first
cellular
ligand
p30
yeast
two-hybrid
assay
plasmids
pgbt9
pact2
bd
sciences
used
sources
gal4
dna-binding
domain
bd
transcriptional
activation
domain
ad
respectively
pgbt9-p30
unrelated
control
protein
pgbt9-p54
independently
used
baits
screen
pact2
cdna
library
from
pig
macrophages
saccharomyces
cerevisiae
reporter
strain
y190
previously
published
18,20,21
yeast
sequentially
transformed
bait
plasmid
pact2
library
lithium
acetate
method
auxotrophic
colony
size
selection
resulting
clones
analyzed
expression
gal4-dependent
β-galactosidase
plasmid
dna
from
clones
exhibiting
β-galactosidase
activity
isolated
retransformed
yeast
strain
y190
pgbt9-p30
eliminate
false
positives
sequence
inserts
determined
sequencing
using
specific
primers
compared
data
base
ncbi
using
blast
program
pgbt9-p30
pgbt9-p54
pact2-k
individually
transformed
yeast
tested
β-galactosidase
activity
exclude
activation
gene
reporter
itselves
identify
cellular
proteins
interacting
asfv
early
protein
p30
yeast
two-hybrid
system
used
screen
porcine
macrophage
cdna
library
selection
from
total
transformants
screened
two
potential
positive
clones
obtained
reporter
gene
assay
dna
sequence
analysis
showed
cdna
contained
clones
identical
size
composition
matched
cdna
sequence
encoding
hnrnp-k
cdna
sequences
from
positive
clones
represent
nucleotides
from
hnrnp-k
cdna
sequence
genebank
accession
number
nucleotide
identity
corresponding
amino
acid
residues
hnrnp-k
protein
addition
diverse
truncations
hnrnp-k
performed
attending
previously
well
characterized
functional
domains
tested
interaction
using
yeast
two-hybrid
system
results
showed
none
three
different
p30
truncations
interacted
hnrnp-k
fig.
2a
other
hand
determine
hnrnp-k
fragment
from
amino
acid
residue
contained
interacting
region
p30
fig.
2b
using
yeast
two-hybrid
system
identified
cellular
hnrnp-k
interacting
protein
asfv
early
protein
p30
schematic
representation
diverse
p30
truncations
tested
interaction
full
length
hnrnp-k
yeast
two-hybrid
assay
schematic
representation
diverse
hnrnp-k
truncations
tested
interaction
complete
p30
yeast
two-hybrid
assaybait-containing
sequence
encoding
acbp4
constructed
yeast
two-hybrid
screens
using
cdna
library
derived
from
a.
thaliana
identify
proteins
interact
directly
acbp4
two-hybrid
library
screens
performed
saccharomyces
cerevisiae
strain
ypb2
mata
ara3
his3
ade2
lys2
trp1
leu2
canr
gal4
gal80
lys2
gal1-his3
ura3
gal1uas17mers
lacz
kohalmi
et
al.
cotransformants
plated
synthetic
dextrose
agar
plates
lacking
leucine
tryptophan
histidine
sd-leu-trp-his
supplemented
3-at
kohalmi
et
al.
s.
cerevisiae
strain
ypb2
transformed
bait
plasmid
pat188
transformants
plated
synthetic
dextrose
agar
plates
lacking
leucine
sd-leu
aliquot
transformants
also
tested
sd-leu-his
medium
supplemented
3-amino-1
4-triazole
3-at
absence
growth
medium
confirm
db
bait
fusion
protein
unable
initiate
transcription
his3
subsequently
bait-carrying
strain
tested
negative
β-galactosidase
activity
using
x-gal
5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside
colony
filter
assay
showed
bait
able
activate
transcription
lacz
reporter
gene
prey
vector
pbi-771
variant
ppc86
chevray
nathans
kohalmi
et
al.
introduced
strain
inability
grow
sd-leu-trp-his
medium
supplemented
3-at
lack
β-galactosidase
activity
confirmed
before
bait
used
cdna
library
screening
ensure
sufficient
coverage
identification
potential
proteins
interacting
acbp4
yeast
two-hybrid
screenings
also
performed
molecular
interaction
facility
university
wisconsin
madison
using
yeast
strains
vectors
previously
described
james
et
al.
bait
preparation
acbp4
amino
acids
cloned
in-frame
gal4
dna-binding
domain
bait
vector
pbute
kanamycin-resistant
version
gal4
bait
vector
pgbduc1
resulting
vector
subject
dna
sequence
analysis
confirm
presence
in-frame
fusion
before
use
transformation
s.
cerevisiae
mating
type
strain
pj69-4a
followed
testing
autoactivation
β-galactosidase
reporter
gene
yeast
ypb2
transformed
bait
gal4
db
acbp4
grow
sd-leu-his
tested
negative
x-gal
colony
filter
assays
data
shown
suggesting
pat188
bait
alone
activate
transcription
reporter
genes
his3
lacz
deemed
appropriate
two-hybrid
screens
gal4
ta
tagged
a.
thaliana
cdna
library
introduced
yeast
ypb2
harbouring
plasmid
pat188
number
independent
transformants
determined
following
transformation
plating
aliquot
yeast
transformation
mixture
sd-leu-trp
total
putative
positives
selected
sd-leu-trp-his
supplemented
3-at
medium
putative
positives
screened
β-galactosidase
activity
using
x-gal
colony
filter
assay
nine
yeast
clones
appeared
blue
varying
intensities
identified
putative
clones
encoding
interactors
putative
library
plasmids
retrieved
nucleotide
sequences
searched
against
blast
server
http://www.ncbi.nlm.nih.gov/cgi-bin/blast
only
one
clone
in-frame
gal4
ta
encoding
full-length
ethylene-responsive
element
binding
factor
erf
protein
atebp
arabidopsis
genome
locus
at3g16770
ap2/erebp
ethylene-responsive
element
binding
protein
domain
present
atebp
amino
acids
okamuro
et
al.
another
independent
yeast
two-hybrid
screen
using
molecular
interaction
facility
university
wisconsin
madison
six
putative
positives
identified
following
selection
histidine
drop-out
β-galactosidase
assays
subsequently
used
retransform
yeast
mating
type
strain
pj69-4a
validated
mating
selection
assays
using
acbp4
bait
empty
bait
vector
unrelated
baits
five
clones
tested
positive
identified
nucleotide
sequence
analysis
results
from
analysis
using
blast
revealed
only
one
clone
in-frame
encoded
full-length
actin-depolymerizing
factor
adf3
at5g59880
protein
results
x-gal
filter
assays
shown
fig.
1a
positive
protein
protein
interaction
results
activation
reporter
gene
β-galactosidase
yeast
cells
turns
yeast
colonies
blue
filter
assays
using
x-gal
without
interaction
yeast
colonies
remain
colourless
shown
fig.
1aa
gal4
db
acbp4
fusion
interacted
gal4
ta
atebp
indicated
blue
colour
arising
from
production
significant
levels
β-galactosidase
interactions
observed
control
yeast
cells
harbouring
gal4
db
acbp4
gal4
ta
fig.
1ab
gal4
db
gal
ta
atebp
fig.
1ac
therefore
from
yeast
two-hybrid
analysis
atebp
identified
putative
protein
interacts
acbp4
colony
filter
β-galactosidase
assays
candidate
proteins
atebp
from
yeast
two-hybrid
screens
ypb2/gal4
db
acbp4
gal4
ta
atebp
ypb2/gal4
db
acbp4
gal4
ta
ypb2/gal4
db
gal
ta
atebp
kelch-motif
containing
acbp4
used
bait
yeast
two-hybrid
screens
from
interactor
atebp
retrieved