assess
whether
melk
role
mammary
carcinogenesis
knocked
down
expression
endogenous
melk
breast
cancer
cell
lines
using
mammalian
vector-based
rna
interference
furthermore
identified
long
isoform
bcl-g
bcl-gl
pro-apoptotic
member
bcl-2
family
possible
substrate
melk
pull-down
assay
recombinant
wild-type
kinase-dead
melk
investigate
biological
functions
melk
breast
cancer
cells
searched
substrates
melk
cancer
cells
vitro
protein
pull-down
assays
using
wild-type
melk
wt-melk
kinase-dead
melk
d150a-melk
recombinant
proteins
comparison
silver
staining
sds-page
gels
containing
pulled-down
proteins
identified
approximately
kda
protein
lane
corresponding
proteins
pulled-down
wt-melk
corresponding
proteins
pulled-down
d150a-melk
figure
3a
furthermore
demonstrated
his-tagged
wt-melk
pull-down
bcl-gl
his-tagged
d150a-melk
indicating
vitro
bcl-gl
interacts
directly
wt-melk
d150a-melk
figure
3d
thus
investigate
biological
significance
melk
breast
cancer
cells
searched
possible
substrate
melk
means
vitro
pull-down
assays
recombinant
wild-type
melk
wt-melk
kinase-dead
melk
d150a-melkmembrane
fraction
adult
brain
lysates
incubated
without
flag-tagged
cdk5
flag-tagged
cdk5
pulled
down
trkb
from
membrane
fraction
adult
brain
lysates
quality
purified
gst
gst-fusion
proteins
used
gst
pull-down
assay
verified
coomassie
blue
staininggst-pull
down
co-immunoprecipitation
assays
performed
characterize
interaction
gst-pull
down
assay
identify
region
brca1
interacting
pp1
fragments
used
gst
pull
down
assays
br
diagrammed
gel
depicting
co-precipitation
gst-bound
br-4
pp1
following
incubation
gst-brca1
proteins
equal
amounts
cell
lysate
western
blot
performed
probed
antibody
catalytic
region
pp1
analysis
effect
mutations
kvtf
pp1
interacting
domain
brca1
pp1
interaction
gst-bound-br4
v-a
gst-bound-br4
f-a
binds
pp1
decreased
intensity
compared
wt
gst-bound-br4study
appl1
rab5
interaction
solution
performed
glutathione
s-transferase
gst
mediated
pull-down
assays
appl1
bar-ph
domain
residues
longer
fragment
40-residue
extension
downstream
ph
domain
appl1
each
effectively
pulled
down
gtp-bound
gst
rab5
fusion
protein
figure
appl1
protein
pulled
down
either
wt
rab5
preloaded
non-hydrolysable
gtp
analog
gppnhp
rab5-q79l
defective
gtp
hydrolysis
without
preloaded
gtp
analog
effectively
pulled
down
either
wt
rab5
preloaded
gdp
rab5-s34n
defective
gtp
binding
figure
data
shown
therefore
tested
appl1
binding
specificity
towards
other
members
rab5
subfamily
using
gst
rab21
full
length
gst
rab22
pull
down
appl1
other
hand
unable
detect
binding
between
appl1
rab22
pull-down
assay
figure
define
rab5
appl1
binding
mode
performed
extensive
pull-down
analyses
between
variants
rab5
appl1
looking
reversal
mutants
rescue
lost
binding
ability
others
identified
one
such
pair
appl1-n308d
abolished
binding
rab5
rab5-l38r
effect
appl1
binding
however
rab5-l38r
found
bind
appl1-n308d
other
tested
appl1
variants
similar
hydrophobic-to-charged
mutations
including
v25d
a318d
l321d
supplementary
figure
result
suggests
rab5-l38r
restores
binding
appl1-n308d
complementary
electrostatic
yet
specific
interactions
implies
position
strand
appl1
ph
domain
vicinity
position
helix
rab5
complex
combined
results
from
mutagenesis
pull-down
experiments
figures
crystal
structures
bar-ph
domain
appl1
figure
structures
gtpase
domain
human
rab5
different
nucleotide
binding
modes
zhu
et
al
clearly
explain
requirement
gtp-bound
rab5
appl1
binding
based
available
information
modeled
interaction
between
two
proteins
assumption
both
proteins
remain
rigid
bodies
complex
model
satisfies
constraints
imposed
mutagenesis
pull-down
results
figure
rab5
appl1
pull-down
experiment
gst
rab
fusion
protein
kda
incubated
slurry
gsh
sepharose
4b
ge
healthcare
min
nucleotide
loading
reaction
performed
gsh
beads
exchange
buffer
pbs
dtt
mgcl2
edta
gppnhp
gdp
min
increasing
magnesium
ion
concentration
terminated
loading
reaction
pull-down
analysis
appl1-rab
interaction
gst
fusion
proteins
rab5
rab21
rab22
used
pull
down
his-tagged
appl1
fragments
presence
gdp
gtp
analog
gppnhp
pull-down
assay
rab5
variants
point
mutations
switch
regions
introduced
full-length
rab5-q79l
background
gst
fusion
construct
each
mutant
expressed
e.
coli
purified
equal
amounts
each
rab5
sample
used
pull
down
recombinant
proteins
his-tagged
wt
appl1
top
panel
his-tagged
rabaptin5
bottom
panel
results
visualized
coomassie
blue
stainconsistent
prior
reports
failed
observe
stable
direct
interaction
between
purified
st
pp2a
subunit
using
glutathione
s-transferase
gst
pull-down
assay
unpublished
datanext
used
recombinant
purified
mrn
complex
peptide
pull-down
experiments
showed
mrn
bound
phosphorylated
unphosphorylated
form
sdtd
peptide
confirming
direct
nature
interaction
yet
bind
either
version
h2ax
c-terminal
peptide
fig
3a
indicates
specificity
mrn
phosphorylated
mdc1
sdtd
motif
also
shows
previously
reported
nbs1
binds
γh2ax
directly
kobayashi
et
al
interaction
must
less
stable
mrn
binding
phosphorylated
sdtd
motif
silver-stained
sds
polyacrylamide
gel
sdtd
peptide
pull-down
pp
s329
t331
doubly
phosphorylated
peptide
non-phosphorylated
equivalent
bead-interacting
proteins
removed
from
extracts
pre-clearing
step
molecular
weight
markers
ng
portion
ck2-phosphorylated
mock-phosphorylated
gst-sdtd6
incubated
pull-down
reactions
vitro-translated
ivt
ha
fusions
corresponding
amino-acid
residues
nbs1
ha-fnbs1
analogous
proteins
bearing
point
mutations
predicted
abolish
either
fha
r28a/h45a
brct2
k160m
phosphorylation-dependent
interactionsglutathione
s-transferase
gst
pull-down
experiments
plasmids
pgex-rnp-k
kindly
provided
dr.
levens
pgex-4t
ge
healthcare
used
express
gst-hnrnp-k
fusion
protein
gst
alone
escherichia
coli
gst-hnrnp-k
gst
proteins
produced
e.
coli
bl21
cells
previously
transformed
vector
pgex-rnp-k
pgex-4t
cells
induced
iptg
c.
bacteria
harvested
suspended
lysis
buffer
pbs
triton
x-100
pmsf
dtt
anti-proteases
sonicated
ice
gst-hnrnp-k
gst
alone
purified
from
cleared
lysates
mixing
glutathione-sepharose
4b
beads
ge
healthcare
cleared
lysate/400
beads
c.
extensive
washing
gst-hnrnp-k
gst
beads
incubated
binding
buffer
hepes
ph
nacl
nonidet
p-40
protease
inhibitor
mixture
roche
molecular
biochemical
insect
cell
extracts
containing
either
p30
p54
asfv
proteins
overexpressed
baculovirus
system
equal
amounts
gst
gst-hnrnp-k
asfv
proteins
p30
p54
used
judged
coomasie
blue
staining
interaction
confirmed
vitro
binding
assays
using
gst-hnrnp-k
fusion
protein
bound
glutathione-sepharose
4b
beads
gst
pull-down
experiments
carried
out
followed
western
blot
specific
antibodies
first
p30
another
unrelated
asfv
protein
case
p54
negative
control
used
bind
gst
fusion
protein
gst
alone
protein
p30
p54
retained
presence
gst-hnrnp
showing
band
appropriate
size
kda
p30
western
blotting
band
appear
presence
gst
alone
indicating
specific
interaction
p30
hnrnp
gst
fig.
1a
identical
results
obtained
subsequent
experiments
using
ba71v
infected
mock-infected
cells
extracts
instead
baculovirus
infected
cell
extracts
pull-down
assay
fig.
1b
interaction
confirmed
vitro
gst-fusion
pull-down
assay
using
either
p30
obtained
from
baculovirus
system
asfv
infected
cell
extracts