hela
cells
transiently
co-transfected
hours
plasmid
constructs
encoding
flag-tagged
full-length
bcl-gl
series
flag-tagged
partial
bcl-gl
proteins
fl
n-1
n-2
n-3
n-4
c-1
c-2
c-3
well
amount
plasmid
encoding
ha-tagged
wt-melk
using
fugene6
transfection
reagent
roche
cells
lysed
lysis
buffer
described
above
lysates
pre-cleaned
normal
mouse
igg
rec-protein
sepharose
4b
zymed
san
francisco
ca
usa
minutes
subsequently
lysate
incubated
anti-flag
agarose
m2
gel
sigma-aldrich
hours
washing
three
times
lysis
buffer
proteins
beads
eluted
sds
sample
buffer
validate
interaction
between
wt-melk
bcl-gl
constructed
plasmids
designed
express
ha-tagged
wt-melk
ha-wt-melk
flag-tagged
bcl-gl
flag-bcl-gl
plasmids
co-transfected
hela
cells
proteins
immunoprecipitated
anti-flag
antibody
immunoblotting
precipitates
using
anti-ha
antibodies
indicated
flag-bcl-gl
co-precipitated
ha-wt-melk
figure
3c
determine
segment
bcl-gl
interact
wt-melk
performed
co-immunoprecipitation
analyses
using
ha-wt-melk
partial
bcl-gl
proteins
tagged
flag
figure
3e
co-transfection
plasmid
clones
hela
cells
performed
immunoprecipitation
anti-flag
antibody
immunoblotting
anti-ha
antibody
interaction
melk
bcl-gl
extracts
from
hela-cells
transfected
ha
hemagglutinin
tagged
wt-melk
ha-wt-melk
flag-tagged
bcl-gl
flag-bcl-gl
combination
harvested
hours
transfection
cell
lysates
immunoprecipitated
anti-flag
m2
antibody
precipitated
proteins
separated
sds-page
western
blotting
analysis
performed
anti-ha
antibody
determination
wt-melk
binding
regions
bcl-gl
immunoprecipitation
ha-tagged
wt-melk
various
peptide
sequences
flag-tagged
bcl-gl
figure
3e
pulled
down
immunoprecipitation
flag-m2
antibody
immunoblotted
rabbit
anti-flag
antibody
expression
ha-tagged
wt-melk
total
cell
lysates
confirmed
western
blotting
analysis
control
immunoprecipitation
performed
from
cells
co-transfected
pcaggsn3fc
mock
ha-tagged
wt-melk
ha-wt-melk
stepspp1
isoforms
encoded
different
genes
conserved
across
catalytic
domains
distinct
roles
each
isoform
yet
determined
coimmunoprecipitated
flag-epitope
tagged
pp1α
brca1
observed
isoforms
interacted
brca1
additionally
identified
functional
pp1
interacting
domain
within
brca1
domain
found
other
pp1
regulatory
proteins
suggesting
brca1
may
regulate
activity
pp1
act
scaffold
protein
promote
dephosphorylation
brca1
associated
proteins
pp1co-immunoprecipitation
assays
used
confirm
protein
protein
interactions
subsequently
localization
acbp4
interacting
protein
atebp
confirmed
using
transient
expression
gfp
dsred-tagged
fusion
proteins
nicotiana
tabacum