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Transfection of Caco-2 Cells
For Liter
With
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siR
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NA Usin
lsg_liter
g the
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siL
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equest@bio-r
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ad.com
ipid Reagent
Transfection of Caco-2 Cells W
www
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Lipid Reagent
The full length version of this protocol, authored by Daniel R Clayburgh and
Results and Discussion
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The full length version of this protocol, authored by Daniel R.
Introduction
Results and Discussion
siRNA-mediated knockdown of MLCK1 in Caco-2 cells was
6
Clayburgh
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from cells transfected with either nonspecific control siRNA or
Tight junctions between intestinal epithelial cells are an important
siRNA-mediamea
M
su
L
r
C
e
K
d
1
b
-
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s
ted
p
s
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mknockdo
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s
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i
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ita
(
t
F
of iv
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eMLCK1
u
r
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A
sin
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M
Caco-2 tra
L
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with eit
transcription
her nonspecifi
PCR
c co
of
ntr
RNA
ol siR
from
ntent
NA or
cells
of
T
Rad (bulletin 5370).
component of the permeability barrier separating the potentially
ight junctions between intestinal epithelial cells are an important
transfected
monolay
MLCK1-spe
with
ers t
cific
either
ransfe
siRNA
nonspecifi
cted with
(Figure 1


A
c
co
)
control
ntrol si
. MLCK
siRNA
RNA w
1 mRN
or
as
A
MLCK1-specifi
52% of the t
content of

o
c
tal
harmful contents of the intestinal lumen from the internal milieu.
component of the permeability barrier separating the potentially
siRNA
ML
monola
(F
C
y
igure
K exp
ers tr
1A).
ress
ansf

e
MLCK1
ed. MLC
cted w
mRNA
K1 mR
ith con
content
NA was
trol siRN
of
re
A
monolayers
duced to 27
was 52% of
transfected
% of total
the total
h
Introduction
Cultured monolayers of polarized epithelial cell lines, such as
armful contents of the intestinal lumen from the internal milieu.
with
M
MLCK
control
LCK m
expres
siRNA
RNA, a
sed. M
was
47%
LCK
52%
redu
1 mR
of
cti
NA
the
on,
wa
total
in mo
s red
MLCK
nolaye
uced t
expressed.
rs transfec
o 27% of t

t
o
MLCK1
ed with
tal
C
Tight
MD
ultur
junctions
CK, T84,
ed monola
between
and Caco
yers of p
intestinal
-2, are im
olarized e
epithelial
portant m
pithelial c
cells
odel
ell lin
are
sys
es, s
an
tem
uch
important
s for the study
as
mRNA
ML
MLCK
was
CK1-
mRN
reduced
specific
A, a 47%
to
siR
re
27%
NA. M
duct
of
L
ion
total
CK2
, in m
MLCK
mRNA
onola
mRNA,
conten
yers tr

a
a
t
n
47%
was u
sfecte
reduction,
nchanged
d with

by
component of tight juof ncthe tion permeability structure andbarrier functioseparan. The ting recethe nt depotentially velopment of
MDCK, T84, and Caco-2, are important model systems for the study
in
ML
monolayers
the MLCK1-
CK1-specific
transfected
specific siRN
siRNA. MLC
with
A.
K2 mR
MLCK1-specifi
NA content w
c
as
siRNA.
uncha

n
MLCK2
harmful
siRNA
contents
technolo
of
gy
the
pro
intestinal
vides a ne
lumen
w meth
from
od of
the
pro
internal
bing tigh
milieu.
t junct

ion mRNA content was unchanged by the MLCK1-specifi c siRNA.
ged by
o
Cultured
f ti
r
g
e
h
g
t
u
ju
la
n
monolayers
to
ct
r
i
y
o

n
p

a
s
t
t
h
ru
w
c
a
t
y
u
of
s
re
i

n
a
polarized

n
ce
d
l

l
f

u
m
n
o
c
d
ti
e
o
epithelial
l
n
s
.
.


T
U
h
n
e
fo
r
r
e
tu
c
cell
e
n
n
a
t
t

e
d
lines,
ly
e
,
v
t
e
h
l
e
o


p
such
C
m
ac
e
o
n
-
t
2
as
o

f
cell
the
W
M
e
L

C
c
K
o
1
n
-

s
rm
pe
e
c
d
ifi
t
c
h

a
s
t
iR
s
N
iR
A
N
.
A reduced expression of MLCK1 by
s
MDCK,
iRN
li
A
ne
t


T84,
e
is
c

h
re
n
and
l
o
a
l
t
o
iv
g
e
Caco-2,
y
l

y
p

r
r
o
e
v
s
i
i
d
s

e
t
are
a
s
n
a
t
important

t
n
o
e
t
w
ra
m
ns
e
fe
th
c
model
o
ti
d
o

n
o
,
f


m
p
systems
r
a
o
k
b
in
in
g
g
t

h
ti
for
g
e
h
u
t
the

s
j
e
u

n
o
stud
c
f
t

i
s
o
iR
n
y
NA
We
im
confi
mu
rmed
noblott
tha
ing
t
ce
siRNA
ll lysate
reduced
s (Figure
expression
1B). Only ce
of
lls tr
MLCK1
ansfecte
by
d
r
of
eg
tight
u
te
la
c
t
h
o
junction
n
ry
o

l
p
o
a
g
t
y
h

w
te
structure
a
c
y
h
s
n

i
i
c
n
a
c
ll
e
y
l

and
l
d
m
iffi
o
c
function.
d
u
e
lt
ls
.
.
C
U
a
n
c
f
o

o
The
-
r
2
tu
c
n
recent
e
a
l
t
l
e
s
ly
m
,
u
t
development
h
s
e
t

b
C
e
a

c
tr
o
a
-
n
2
s

f
c
e
e
c
of
ll
ted
immunoblotting
We
w
co
it
n
h

t
r
h
m
e
e
s
d
p
t
e
h
cell
c
a
ifi
t
c
s

lysa
i
s
R
i
N
RN
A
tes

A
re
s
(F
d
h
u
igure
o
c
w
e
e
d
d
e
1B).
a
xp
s
r
i

e
g
Only
s
n
s
ifi
io
c
n
cells
a

n
o
t
f


r
M
e
transfected
d
L
u
C
c
K
t
1
io

n
b

y
in M
with
LCK1
l
siRNA
ine
i
i
n
s


s
r
technolog
e
u
l
s
a
p
ti
e
v
n
e
s
ly
io
r
n
y
e

s
b
provides
i
e
st
f
a
o
n
re
t

t
p
o
l

a
a
t
t
r
i
a
new
n
n
g
s
;
f
c
e
method
e
c
l
t
l
i
s
o

n
th
,
e
m
n
of
a
t
k
y
probing
i
p
n
i
g
c

a
th
lly
e


r
u
e
tight
s
q
e
u

i
o
re
f
junction


s
a
iR
w
N
e
A
ek or
the
imm
specifi
p
u
r
n
ot
o
e
b
i

n
l
c
o
.
t

siRNA
T
ti
h
n
i
g
s


c
re
e
sho
d
ll
u
ly
c
wed
s
ti
a
o
t
n
e

s
a
in
(
signifi

F
e
ig
xp
u
r
r

e
e
cant
s
1
s
B
io
)
n
reduction
.

O
h
n
a
l
d
y


a
c

e
s
l
i
l
g
in
s
n
t
MLCK1
i
r

a
c
n
a
s
n
fe
t
c
e
t
protein.
f
e
fe
d
ct on

tregulaech
m
no
o
torl
r
o
e
g

y
o
y
f
pa

t
c
e
u
thways ch
ltu
n
r
ic
e
a
t
l
o
lyin


d
d
e
cell if
v

e
c
lo
umodels.
p
lt

.
f
C
un
a
c
c
t
o
io
-Unfortuna2
n

a
c
l
e
t
l
i
l
g
s
h
m
t
tely
j
u
u
s
n
t
c
, b
t
the
i
e
o

n
tr
s
aCaco-2
.
n
S
s
in
fe
c
c
e
t

e
m
cell d
ost
This
with
t

h
t
reduction
h
e
e
t

i
s
g
p
h
e
t
c
ju
ifi
n
c
in
c

t
s
i
expression
o
iR
n
N
p
A
e

r
s
m
ho
e
w
ab
e
had
i
d
li

t
a
y


s
o
a
i
f
g

signifi
t
n
h
i
e


c
C
a

a
cant
n
c
t
o
r
-
e
2
d
effect

u
m
c
o
ti
n
o
o
n
on
l

a
in
y

e
M
the
rs
L
;
C
c
tight
K
e
1
lls
i
line
n su
is
sp
rela
ens
tively
ion be
resistant
fore platin
to
g;
transfection,
cells then ty
making
pically re
the
qui
use
re a
of
we
siRNA
siRNAs have a half-life within cells of less than 4 days, tra
e
d
k
i

t
o
io
r
nal
junction prot
t
e
r
i
a
n
n
.
s
Tpermeability
f
h
e
i
c
s
t

e
re
d
d
w
u
i
c
th
tio
M
nof
L
in
C
the
K
e
1
xp

Caco-2
s
r
i
e
R
s
N
si
A
o

n
e
monolayers;
x
h
h
a
i
d
b

it
a
e

d
si

g
a
n
s
ifi
ig
ccells
n
a
i
n

t
c

a
etransfected ffect on
m
technolog
ore of cu
y
lt
technically
ure to devel
diffi
op

f
cult.
unc

ti
Caco-2
onal tigh
cells
t junc
must
tions.
be
Sin
transfected
ce most
with
the ti
MLCK1
ght junc
siRNA
tion pe
exhibited
rmeabilit
a
y
signifi
of the
cant
Caco
increase
-2 mono
in
la
transepithelial
nt increase
yers; cells
s
in
iR
suspension
methods of
NAs have a
before
Caco-
half-life
pla
2 tr
w
ting;
ansfe
ithin
cells
ction
cells
then
are
of les
typically
not adeq
s than 4
require
uate for
days, tr
a
s
ad
week
iRNA-
itiona
or
l
resistance
in trans
transfected
compared
epithelial re
with MLCK
to
sis
1
those
tance
siRNA
transfected
compared t
exhibited a
with
o tho
signifi
nonspecifi
se transfe
cant incr

e
c
cte
a
siRNA
d with
se
m
more
me
etho
of
dia
ds
culture
ted kno
of Caco
to
ckd
-2
develop
own. Kn
transfect
functional
ockdown u
ion are not
tight
sing
adeq
junctions.
siLentFec
uate for s

t
i
Since
-media
RNA-
most
ted
(F
in
igure
nons
transe
2).
pe
pi

t
The
cific
helia
use
siRN
l res
of
A (
ista
siLentF
Figure
nce co
ect
2).
mp
in
Th
are
conjunction
e use of siLe
d to those tr
with
ntFec
ansfe
high-density
t in conjunction
cted with
m
siRNAs
trans
ediate
ha
fe
d k
ve
ctio
no
a
n
ck
half-life
of siRNA
down. K
within
and h
nockdo
cells
igh-d
wn u
of
en
sin
less
sity p
g siL
than
lating
entFe
4
o
c
days,
f Cac
t-med
traditional
o-2 cells
iated
pla
non
ting
with
spec
allo
hig
ific
wed
h-de
siRN
us
nsit
A (
to
y p
Fig
measure
lating allo
ure 2). Th
the
wed
e u
effects
us to m
se of siL
of
e
en
knockdo
asure the
tFect in
wn
eff
con
on
ect
junc
tight
s
tion
t
methods
allowe
ransfecti
of
d
on
Caco-2
the ear
of siRN
transfection
ly developm
A and high-
are
ent
den
not
of t
sity
adequa
ight jun
plating
te
ct
o
for
ion
f C
siRNA-media
s, permitting
aco-2 cells
ted
the
junction
of kno
with high-
physiolog
ckdown o
density p
y
n
lat
and
tight
ing a
the
junct
llowe
molecular
ion physiolo
d us to me
regula
gy an
asure
tion
d the
the e
of
mo
ffec
intestinal
lecular
ts
knockdostudywn. of tiKnockdoght junctwn ion pusing hysiolosiLentFgy. Witect-mediah this metted hod,transfection we were able
allowed the early development of tight junctions, permitting the
permeability
regulatio
of knockdow
.
n of intestinal permeability.
of siRNA
to inter
and
fere w
high-density
ith expressio
pla
n o
ting
f my
of
osin
Caco-2
light ch
cells
ain ki
allo
nas
wed
e (ML
the
CK)1, a
n on tight junction physiology and the molecular
study of tight junction physiology. With this method, we were able reg

u
A
l

a

t

i

on o
N
f
o

n
in
sp
t
e
e
c
s
ifi
t
c
inal p
M
e
L
r
C
m
K
e
1
abili
B
ty.
Nonspecific MLCK1
early
sp
development
lice variant of
of
ML
tight
CK, a
junctions,
nd to stu

d
permitting
y the effec
the
t on
stud
elec
y
tr
of
oph
tight
ysiology.
t
junction
o interfe
physiolog
re with ex
y
p
. With
ressi
this
on o
method,
f myosin
we
ligh
were
t cha
able
in ki
to
na
interfere
se (MLCK
with
)1, a
A Nonspecific MLCK1 B Nonspecific MLCK1
s
expression
plic
A
e
b
v
b
a
r
r
e
ia
v
n
of
ia
t
t
o
myosin
e
f
d
M
M
L
e
C
t
K
light
h
,
o
a
d
n
s
d
chain
to st
kinase
udy the
(MLCK)1,
effect on
a
e
splice
lectrop
variant
hysiolo
of
gy.
MLCK1
MLCK,
Cac
and to stud
o-2 cells w
y the effect on electrophysiolog
ere plated on permeable suppo
y.
MLCK2
rts. A sequence
MLCK1
Abb
u
r
n
e
i
v
q
i
u
a
e
te
t
d
o

t
M
he
e

t
M
ho
L
d
C
s
K1 splice variant was used to design the siRNA. F
M
ig
L
.
C
1
K
F
.
2
ig. 1. Knockdown of MLCK1 in transfected Caco-2 cells.* A, RT-PCR of
CAbbreviated Methodsac
s
o
i
-
R
2
N
c
A
e
o
lls
r

n
w
o
e
n
r
s
e
p
p
e
l
c
a
i
t

e
c
d


c
o
o
n
n

t
p
r
e
o
r
l
m
si
e
R
a
N
b
A
le
s
s
o
u
lu
p
t
p
io
o
n
rt
s
s

.
w
A
e

r
s
e
e

q
a
u
d
e
d
n
e
c
d
e
Knockdown of MLCK1 in transfected Caco-2 cells.* A, RT-PCR of
to buffer
MLCK
M
s
L
p
C
li
K
c

e
sp
v
l
a
ic
r
e
ia
v
n
a
ts
ria
M
nt
L
s
C
M
K
L
1
C
(
K
u
1
p

p
(u
e
p
r
p
b
e
a
r
n
b
d
a
)
n
a
d
n
)
d
an
M
d
L
M
C
L
K
C
2
K
(
2
lo
(
we
low
r
e
b
r
a
b
n
a
d
nd
)
)
f

r
f
o
ro
m
m Caco-2
u
Caco-2
nique t
cells
o the
were
MLC
pla
K1
ted
spl
on
ice
permeable
variant wa
supports.
s used to
A
d
sequence
esign the
unique
siRNA.
monolayers transfected with nonspecific or MLCK1-specific siRNA. B, immunoblots
C
F
a
i
c
g
o
.
-
1
2
. K
m
n
o
o
n
c
o
k
l
d
ay
ow
ers
n
t
o
r
f
a

n
M
s
L
fe
C
c
K
te
1
d
in
w
t
i
r
t
a
h
n
n
s
o
fe
n
c
s
t
p
e
e
d
c

i
C
fic
ac
o
o
r
-
M
2 c
L
e
C
l
K
ls
1
.*
-

s
A
p
,
e
R
c
T
if
-
i
P
c
C
s
R
iR
o
N
f
A.
s
to
iR
the
con
NA
MLCK1
taining
or nons
splice
siLent
pecific
variant
Fect lip
contro
was
id re
l siRN
used
agent
A so
to
an
lut
design
d allow
ions we
the
ed
re a
siRNA.
to incu
dded

t
siRNA
bate. A
o buffe
or
t
of lysates from monolayers transfected with nonspecific and MLCK1-specific siRNA
the same time, Caco-2 cells were incubated with trypsin. Afte
r
MLCK splice variants MLCK1 (upper band) and MLCK2 (lower band) from Caco-2
r
B
m
,
o
im
no
m
w
lay
i
u
th
n
e

o
M
r
b
s
L

l
t
o
C
r
t
a
K
s
n
1
s
o
-
f
s
f
e
p
l
c
y
e
t
s
e
c
a
d
if
t
i

e
c
w
s
a
it
n
h
fr
t

o
n
is
m
o
e
n
ra
m
s
.
p

o
A
e
n

c
s
o
if
i
l
g
i
a
c
n
y
o
i
e
fi
r
r
c

s
M
an
t
L
r
t
C
a
d
n
K
r
s
1
o
f
-
p
e
s

c
p
in
t
e

e
M
c
d
if
L
i
w
c
C

i
K
s
th
i
1
R

n
N
m
o
A
R
n
.
N

s
B
p
A
,
e


i
a
m
c
n
if
m
d
ic

u
p
a
n
rot
n
o
d
b
e
l
i
ot
n
s
c
nonspecifi
ontaining
c
s
control
iLentFe
siRNA
ct lipid
solutions
reagent
were
and a
added
llowed
to
to
buffer
incuba
containing
te. At
M
o
L
f
C
ly
K
s
ex
a
1
t
-p
e
sr
s
pe
f
es
ro
scii
m
ofinc
m
is
o
i Ro
n
b
o
Ns
l
A
ay
erw
e
v
r
ei
s
tdh
t

r
iMn
a

n
mL
s
C
f
o
e
Kn
c
1o
te
-lays
d
p

e
w
er
i
cs
th
itf

ri
n
c a
o
na
n
sn
s
fet
p
ics
e
te
c
er
i
d
f
a
i

c
. w

A
a
it
n
hs
d
iM

g
M
nL
L
iCf
C
iKc
K
1a
1
-ns
-
t
s
pd
p
er
e
co
c
ifp
i
i
f
c
ic
i n s
s
iR
iR
N
N
A
A
.
t
siLentF
tryp
he sam
ect
sini
e ti
lipid
zation
me, C
rea
, th
ac
gent
e ce
o-2 c
and
lls w
ells
allo
ere
we
wed
resu
re inc
to
sp
ub
incuba
ended.
ated w
te.
Th
ith
At
e w
try
the
ash
psi
same
ed su
n. Afte
time,
spen
r

sion
MwLitChK M1 L
1
mC
40
KR1N-A spaencd ificp raontteisin ereax. pAr essigsnioifn icais nto dbrsoepr vine d MiLn CmK1o nmoRlaNyAer as ntdra pnrsotfecin ted
was added to previously prepared transfection mixes. The cell w
ex
ith
pr
M
es
L
s
C
io
K
n
1
i
-
s
s
o
p
b
e
s
c
e
if
r
i
v
c
e
s
d
i

R
in
N
m
A
o
.
nolayers
F
tr
i
a
g
n
.
s
2
f
.
e
N
ct
o
e
r
d
m
w
a
i
l
t
i
h
z

e
M
d
L
T
C
E
K
R
1-

s
in
p

e
C
c
a
if
c
ic
o

-
s
2
iR
m
N
o
A
n
.
olayers
t
Caco-2
rypsiniz
cells
ation,
were
the c
incuba
ells we
ted
re re
with
susp
tr
e
ypsin.
nded.


After
The w
tr
a
ypsiniza
shed su
tion,
spen
the
sion
transfected with nonspecific and MLCK1-
cells
su
were
spens
resuspended.
ion and siRNA
The
mixe
washed
s were th
suspension
en plated a
was
t high
added
densit
to
y and 140
was added to previously prepared transfection mixes. The cell
Fi
F
g
ig
.
s
.
2
p
.
2.
N
ec
o
i
N
r
f
o
m
ic
rma
a
s
l
i
i
R
z
li
e
N
ze
d
A
d
T
.*
T
E
T
E
R
E
R

R
in
w
in
C
a
C
a
s
a
c
n
c
o
o
o
-
r
2
m
-2
m
al
m
o
iz
o
n
e
n
o
d
o
l

a
to
120
yers
previously culturedprepared for 4 daytransfection s to allow tigmixes.ht junc tThe ion acell ssemsuspension bly and poland arization
trans
m
fe
o
c
n
t
o
e
lay
d w
er
i
s
t

h
tr
n
an
o
s
n
f
s
e
p
ct
e
e
c
d
i

f
w
ic
i

t
a
h
n
n
d
o

n
M
s
L
p
C
ec
K
i
1
fic
-
siRNA.
suspension and siRNA mixes were then plated at high density and

T
E
R
laye
T
r
E
s
R
t

r
w
a
a
n
s
s

f
s
e
i
c
g
t
n
e
if
d
ic
w
an
it
t
h
ly
n
h
n
o
ig
o
n
h
r
s
m
e
p
r
a
e
i
l
n
c
iz

i
m
f
e
i
d
c
o

n
to
olayers
siRNA
befo
mixes
re use
were
in ele
then
ctrop
pla
hy
ted
siolo
a
g
t
y
high
expe
density
riments.
and
Qua
cultured
ntitation
for
of M
4
LCK1
specific siRNA.* TER was
cultured for 4 days to allow tight junction assembly and polarization
E
R
120
a
l
i
z
e
d
100 m
a
o
n
n
d
o
tr
l
aM
ay
nLs
e
Cf
r
e
s
Kc
t
1t
r
e-
a
ds
n
p
s
we
fe
ict
c
hif
t
i
e
Mc
d
Ls
w
CiR
i
K
t
N
h
1

A-
n
s.
o
p*
n
eT
s
cE
p
ifR
e
ic
c
w
i
s
fi
ia
c
Rs

N
si
A
R
.
N
E
A
r
.
ror bars
days
an
to
d
allo
ML
w
CK
tight
2 mR
junction
NA was
assembly
performe
and
d us
polariza
ing PCR
tion
and
before
weste
use
rn b
in
lotting. o
r
m
TEnRor
r
wma
e
a
p
s
r
l
e
i zs
s
ei
e
gd
n
n
t
tif

o i
s
c
t
ma
a
n
n
ot
d
lny
a
o
r
hl
d
aig

y
e
he
r
e
r
r
o
s r
r
i
.
ntr amnosnfeoclayteed rs with
b
electrophysiolog
efo
E
r
l
e
e

c
u
t
s
ri
e
c

a
in
l

p
e
o
le
t
y
e
c
n
t
experiments.
r
t
o
ia
p
l
h
d
y
i
s
ff
io
e
l
r
o
e
g
n
y
c


e
Quantita
e
s
x

p
w
e
e
ri
r
m
e
e
tion
m
n
e
ts
a
.
of
s
Q
ur
u
MLCK1
e
a
d
n
,
t

i
a
ta
n
t
d
i
and
o
t
n
ra
o
n
MLCK2
f MLCK1 i
z
e
d

T
N
sepithelial
a
l
100
tr
n
an
o
s
n
f
s
e
p
c
e
te
c
d
if

i
w
c
i
s
th
iR
M
N
L
A
C
.
K
T
1
E
-s
R
p
w
ec
a
i
s
fic
s
s
ig
iR
n
N
ifi
A
c
.
a
E
n
r
tl
r
y
or bars
and MLCK2 mRNA was performed using PCR and western blotting.
80
mRNA
resi
was
stanc
performed
e was dete
using
rmined
PCR
usin
and
g Oh
W
m
estern
’s law.
blotting. Electrical
o
r
m
re
h
p
ig
re
h
s
e
e
r
n
i
t
n
st
m
an
o
d
n
a
o
r
la
d
y
e
e
r
r
r
s
or
t
.
N
ransfected with
0
E
potential
lectrical
differences
potential dif
were
feren
measured,
ces were m
and
eas
transepithelial
ured, and trans
resistance
epithelial
80
Nonspecific MLCK1
MLCK1-specific siRNA. Error bars represent
resistance was determined using Ohm’s law.
siRNA siRNA
was determined using Ohm’s law. 0
standard error.
* DaNtaon fsrpoemci fiCclaybMuLrgChK 1DR et al., A differentiation-dependent splice variant of
myosin
si

R
li
N
g
A
ht chain
s
k
iR
in
N
a
A
se, MLCK1, regulates epithelial tight junction permeability,
* Da
t
t
J
a
a


B
f
f
r
r
i
o
o
m
m
l C
C
h
C
l
e
ay
la
m
y
b

b
2
u
u
7
r
r
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h
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h

5
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.
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e
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)
i
.
a
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r
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on
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p
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p
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omf yomsiyno lsigin ht clighhait n kcihnaisn e, kMinLaCsKe1, , rMegLuClaKt1e, s erepguithlealitaels tigehpt ijtuhneclitaiol n tpigehrmt ejuanbcilittiyo, n
p
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e
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r
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,
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506–55513 (2004).
Protocol Guide ı 2008 View entire protocol online at www.biotechniques.com/protocol ı BioTechniques ı 67
BioRad GeneTrans Protocol.indd 67 10/26/07 12:03:49 PM
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