MOLECULAR AND CELLULAR BIOLOGY,
Copyright © 1999, American Society for Microbiology. All Rights Reserved.
Sept. 1999, p. 6318–6322Vol. 19, No. 9
Phenobarbital-Responsive Nuclear Translocation of the
Receptor CAR in Induction of the CYP2B Gene
TAKESHI KAWAMOTO, TATSUYA SUEYOSHI, IGOR ZELKO, RICK MOORE,
KIMBERLY WASHBURN, AND MASAHIKO NEGISHI*
Pharmacogenetics Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of
Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709
Received 16 April 1999/Returned for modification 1 June 1999/Accepted 16 June 1999
The constitutively active receptor (CAR) transactivates a distal enhancer called the phenobarbital (PB)-
responsive enhancer module (PBREM) found in PB-inducible CYP2B genes. CAR dramatically increases its
binding to PBREM in livers of PB-treated mice. We have investigated the cellular mechanism of PB-induced
increase of CAR binding. Western blot analyses of mouse livers revealed an extensive nuclear accumulation of
CAR following PB treatment. Nuclear contents of CAR perfectly correlate with an increase of CAR binding to
PBREM. PB-elicited nuclear accumulation of CAR appears to be a general step regulating the induction of
CYP2B genes, since treatments with other PB-type inducers result in the same nuclear accumulation of CAR.
Both immunoprecipitation and immunohistochemistry studies show cytoplasmic localization of CAR in the
livers of nontreated mice, indicating that CAR translocates into nuclei following PB treatment. Nuclear
translocation of CAR also occurs in mouse primary hepatocytes but not in hepatocytes treated with the protein
phosphatase inhibitor okadaic acid. Thus, the CAR-mediated transactivation of PBREM in vivo becomes PB
responsive through an okadaic acid-sensitive nuclear translocation process.
Hepatic microsomal cytochromes P450 (CYPs) display di-
verse functions in the metabolism of biological signaling mol-
ecules such as steroid hormones and xenochemicals, including
pharmaceutical drugs and environmental contaminants. Induc-
ible gene transcription by exposure to xenochemicals is char-
acteristic for CYPs and increases the organism’s metabolic
capabilities against chemical toxicity and carcinogenicity (3).
Phenobarbital (PB) is the prototype for a large number of
structurally diverse xenochemicals that induce CYPs and other
xenochemical-metabolizing enzymes (3, 10, 20). The PB-re-
sponsive enhancer module (PBREM), a versatile enhancer
capable of responding to numerous PB-type inducers, regu-
lates PB induction of the CYP2B genes in mouse, rat, and
human cells (8, 11, 13, 18, 19). PBREM contains two DR-4
nuclear receptor-binding motifs, NR1 and NR2. Acting as a
retinoid X receptor (RXR) heterodimer, the liver-enriched
constitutively active receptor (CAR) increases its binding to
NR1 in PB-treated mice. Moreover, CAR can stimulate tran-
scriptional activity from a cis-linked PBREM-containing re-
porter plasmid transfected into HepG2 cells (11). CAR-medi-
ated transactivation, however, is constitutive in HepG2 cells,
and the remaining key question is how CAR responds to PB in
inducing the transcription of the CYP2B gene in the liver.
CAR was originally characterized as a constitutive activator
of an empirical set of retinoic acid response elements (1).
Forman et al. have recently shown that this activity can be
repressed by 3?-androstenol in transfected CV-1 and HepG2
cells (7). These results suggest the presence of ligands which
may act positively to confer a regulatory capability to CAR (7,
15). We have demonstrated that 3?-androstenol represses ex-
pression of the endogenous CYP2B6 gene in stable HepG2
cells transfected with a CAR-expressing plasmid. Moreover,
PB activates (i.e., induces) the repressed CYP gene (19). Sim-
ilarly, 3?-androstenol-repressed PBREM can be reactivated by
PB in HepG2 cells. An activation by PB of repressed CAR may
be a mechanism regulating the induction of CYP2B genes.
Despite the fact that binding of CAR to PBREM depends on
PB treatment in vivo, an in vitro-translated CAR-RXR het-
erodimer binds to PBREM without the presence of PB (11).
Moreover, 3?-androstenol does not directly interfere with the
ability of CAR to form a dimer with RXR or to bind to the
elements (7). Thus, an additional or alternative mechanism
that confers PB responsiveness to CAR may remain undetec-
Binding of CAR to NR1 occurs only after PB induction in
liver in vivo (11), indicating that the function of CAR in the
liver may differ from that in HepG2 cells. To explore this
possibility, we examined the intracellular localization of CAR
in mouse liver and primary hepatocytes. Nuclear receptors
generally reside in nuclei and can be activated upon ligand
binding. Constitutively active CAR may be excluded from nu-
clei to suppress unwanted gene activation in nontreated mice.
If, in fact, CAR localizes to liver nuclei following PB treat-
ment, the induction of CYP2B genes may be regulated through
a nuclear translocation process. Western blot and immunohis-
tochemistry studies have been applied to demonstrate the cy-
toplasmic localization of CAR in nontreated mice. CAR un-
dergoes nuclear translocation in livers of PB-treated mice.
PB-induced nuclear translocation appears to be regulated
through a phosphorylation-dephosphorylation pathway. More-
over, various PB-type inducers have been tested to see whether
nuclear translocation of CAR is a general mechanism involved
in CYP2B induction.
MATERIALS AND METHODS
Plasmids. To construct the green fluorescent protein (GFP)-CAR expression
plasmid, CAR cDNA was cloned into the pEGFP-C1 vector (Clontech).
(NR1)5-tk (thymidine kinase)-luciferase plasmid was constructed by cloning
quintuple NR1 sequences in front of the tk-luciferase promoter (at the BglII site)
as described previously (19).
* Corresponding author. Mailing address: Laboratory of Reproduc-
tive and Developmental Toxicology, National Institute of Environmen-
tal Health Sciences, National Institutes of Health, Research Triangle
Park, NC 27709. Phone: (919) 541-2404. Fax: (919) 541-0696. E-mail:
Transfection assays. HepG2 cells were cultured in minimal essential medium
supplemented with 10% fetal bovine serum. (NR1)5-tk-luciferase plasmids (0.1
?g) were cotransfected with GFP-CAR expression plasmids (0.2 ?g) and pRL-
SV40 (0.1 ?g) into HepG2 cells by calcium phosphate coprecipitation. Mouse
primary hepatocytes were prepared from 2-month-old Cr1:CD-1(ICR)BR males
by a two-step collagenase perfusion and were cultured as previously described
(8). (NR1)5-tk-luciferase plasmids (10 ?g) were cotransfected with pRL-SV40 (3
?g) into primary hepatocytes by electroporation. Luciferase activity was mea-
sured by using the Dual-Luciferase reporter assay system (Promega). To visual-
ize the GFP-CAR fusion protein, HepG2 cells transfected with GFP-CAR ex-
pression plasmids were fixed with 4% paraformaldehyde and nuclei were stained
with Hoechst S33258. Intracellular localization of GFP-CAR was determined by
classifying GFP fluorescence-positive cells into three different categories under a
fluorescence microscope: N ? C (nucleus-dominant fluorescence) N ? C (equal
distribution of fluorescence in cytoplasm and nucleus), and N ? C (cytoplasm-
Liver nuclear extracts and DNA affinity chromatography. Forty Cr1:CD-1
(ICR)BR males were treated by intraperitoneal injection with PB (100 mg/kg) or
various PB-type inducers: 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP;
0.3 mg/kg), chlorpromazine (CPZ; 50 mg/kg), 1,1,1-trichloro-1,2-bis(o,p?-chloro-
phenyl)ethane (o,p?-DDT; 1 mg/kg), and 2,3,3?,4?,5?,6-hexachlorobiphenyl (PCB;
3 mg/kg). Subsequently, liver nuclear extracts were prepared from 10 mice at
each time point after induction as described previously (11). For affinity purifi-
cation of CAR, 1 mg of the nuclear extracts was incubated with NR1-conjugated
Dynabeads as previously described (11). The bound proteins were eluted with
100 ?l of 0.5 M NaCl from the beads, and 30-?l aliquots of the elutes were
subjected to Western blot analysis.
Whole-liver extracts and immunoprecipitation. A mouse liver was homoge-
nized with 5 ml of 10 mM Tris-HCl buffer (pH 7.5) containing 0.2 mM sodium
orthovanadate, 1% Triton X-100, 0.5% Nonidet P-40, and 0.2 mM phenylmeth-
ylsulfonyl fluoride. After incubation for 30 min at 4°C, the homogenate was
centrifuged at 50,000 ? g for 30 min at 4°C to obtain whole-cell extracts.
Anti-CAR antibody was enriched from rabbit antiserum raised against recom-
binant human CAR by chromatography using recombinant mouse CAR as the
affinity ligand. For immunoprecipitation, 1 ml of whole-cell extracts or 50 ?g of
nuclear extracts was incubated with anti-CAR antibody for 1 h at 4°C and with
protein A-Sepharose (Pharmacia) for 1 h at 4°C. Then, the Sepharose was
recovered by centrifugation at 1,000 ? g for 1 min, washed with homogenizing
buffer three times, and subjected to Western blot analysis.
Primary hepatocytes. Mouse primary hepatocytes were pretreated with oka-
daic acid (OA; 10 nM) for 30 min and then induced by PB (1 mM), TCPOBOP
(50 nM), or the solvent (dimethyl sulfoxide) for 90 min. The nuclear extracts
were prepared from these hepatocytes by the method of Dignam et al. (6). Total
RNAs were extracted from the hepatocytes for Northern blot analysis at 9 h after
PB induction, using TRIZOL reagent (Life Technologies).
Western blot, Northern blot, and gel shift assays. Nuclear extracts were
resolved on a sodium dodecyl sulfate–10% polyacrylamide gel, transferred to a
polyvinylidene difluoride membrane, and incubated with anti-CAR or anti-
RXR? antibody (Santa Cruz Biotechnology). After incubation with the second-
ary anti-rabbit immunoglobulin G (IgG)-horseradish peroxidase conjugate, the
immunoreactive bands were visualized with an enhanced chemiluminescence
system (Amersham). For Northern blot analysis, 20-?g aliquots of RNAs were
separated on a 1% agarose gel containing 2.2 M formaldehyde, transferred to a
nylon membrane, and hybridized separately with either a 360-bp Cyp2b10 or
180-bp albumin cDNA probe as described previously (8). Gel shift assays were
performed as described previously (11). Reverse transcription-PCR was per-
formed to measure CAR mRNA, using AmpliTaq Gold DNA polymerase (Per-
kin-Elmer Cetus) and the specific primers 5?-TCTCACTCAACACTACGGTT
C-3? and 5?-TCAACTGCAAATCTCCCCGA-3?.
Immunohistochemistry. The paraffin-embedded liver from nontreated and
PB- or TCPOBOP-treated (for 3 h) mice was fixed with 4% paraformaldehyde
for 6 h. Sections (5 to 7 ?m thick) were deparaffinized and blocked with goat
serum. Colorimetric detection was performed by the Vectastain protocol (Elite-
ABC kit; Vector Laboratories), using anti-CAR antibody.
RESULTS AND DISCUSSION
Different regulation of CAR in HepG2 and primary hepato-
cytes. As expected from our previous findings (19), expression
of the transfected (NR1)5-tk-luciferase was activated by co-
transfection of HepG2 cells with the CAR expression vector
(Fig. 1A). 3?-Androstenol repressed this CAR-mediated acti-
vation, whereas the potent PB-type inducer TCPOBOP dere-
pressed (NR1)5-tk-luciferase activity. In sharp contrast, the
transfected (NR1)5-tk-luciferase gene was not activated in con-
trol murine hepatocytes unless the hepatocytes were treated
with TCPOBOP (Fig. 1B). Noticeably, 3?-androstenol treat-
ment did not affect (NR1)5-tk-luciferase activity in hepato-
cytes. To determine how CAR was regulated in HepG2 cells,
we examined the intracellular localization of GFP-CAR in
HepG2 cells (Fig. 1A). The expressed GFP-CAR was always
localized in the nuclei of HepG2 cells, suggesting that CAR
spontaneously translocated to nucleus. Moreover, the treat-
ment with either 3?-androstenol or TCPOBOP did not alter
the nuclear localization of GFP-CAR in HepG2 cells. Because
NR1 was inactive in the absence of TCPOBOP and did not
respond to 3?-androstenol in hepatocytes, the regulation of
CAR may be different from that in HepG2 cells.
Nuclear accumulation of CAR in liver following PB treat-
ment. Binding activity of NR1 to CAR is increased extensively
by PB treatment but is very low in liver nuclear extracts from
nontreated mice (11). We examined whether this increase of
binding in response to PB resulted from the nuclear accumu-
lation of CAR or a functional activation of preexisting CAR.
For this, total liver nuclear extracts were subjected to Western
blot analysis using an affinity-purified anti-CAR antibody. As
shown in Fig. 2A, CAR was barely detectable in liver nuclear
extracts from nontreated mice, but the nuclear content of CAR
was dramatically increased within 1 h after PB treatment. In
sharp contrast, RXR? remained at similar levels before and
after PB treatment (Fig. 2A). Thus, PB treatment resulted in a
specific accumulation of CAR in liver nuclei. An increase of
CAR binding activity to NR1 correlated with that of its nuclear
accumulation, as indicated by Western blot analysis of the NR1
affinity fractions (Fig. 2B). Following the nuclear accumulation
of CAR and its binding to NR1, the Cyp2b10 mRNA began to
increase at 1 h and reached its maximum level 6 h after PB
treatment (Fig. 2C). These induction kinetics were consistent
with our previous findings (11).
Nuclear accumulation in response to various PB-type induc-
ers. It is known that CAR transactivates PBREM in response
to various PB-type inducers (19). Using liver nuclear extracts,
we performed Western blotting to see whether these inducers
also trigger nuclear accumulation of CAR (Fig. 3). TCPOBOP
and CPZ were as effective as PB in producing accumulation of
CAR in liver nuclei. Also, o,p?-DDT and PCB induced accu-
mulation of nuclear CAR, although less effectively than PB.
Increased levels of nuclear CAR accumulation in livers gener-
ally correlated with that of PBREM transactivation in HepG2
cells. This correlation, however, was not perfect; for example,
CPZ transactivated PBREM approximately four times more
effectively than PB in HepG2 cells (19), whereas the two in-
ducers produced similar levels of nuclear CAR in livers. Phar-
macokinetic differences in absorption or metabolism may have
contributed to some of these poor correlations. Nevertheless,
many, if not all, PB-type inducers elicited nuclear accumula-
tion of CAR in mouse livers.
Immunochemical evidence for nuclear translocation in-
duced by PB. To confirm the presence of CAR in nontreated
mouse livers, we performed immunoprecipitation assays of
CAR from whole-liver-cell extracts. Immunoprecipitates were
subjected to Western blot analysis using an anti-CAR antibody
(Fig. 4). An immunostained band that corresponded to CAR
was easily detected in whole-cell extracts of both nontreated
and PB-treated mouse livers. A slight increase of CAR in
whole-cell extracts from PB-treated mice may be within the
range of experimental variation. Thus, the extraction efficiency
of CAR from whole liver cells may have contributed to this
variation. In contrast, but consistent with findings shown in Fig.
2, CAR was immunoprecipitated only from the liver nuclear
extracts of PB-treated mice (Fig. 4). Thus, CAR appeared to
be present as a cytoplasmic receptor in the livers of nontreated
mice and was localized to nuclei following PB treatment. Im-
munohistochemistry was also used to visualize the intracellular
localization of CAR on paraffin-embedded liver sections pre-
VOL. 19, 1999 INDUCIBLE NUCLEAR TRANSLOCATION OF THE RECEPTOR CAR6319
pared from nontreated and PB-treated mice. In PB-treated
mouse livers, CAR was clearly localized in the nuclei (Fig. 5A).
Only a few positively stained nuclei were observed around
vessels in the nontreated liver sections; the majority of nuclei
were devoid of staining compared with the cytoplasmic regions
(Fig. 5B). Taken together, these results lead us to conclude
that PB elicits the nuclear translocation of CAR in liver.
OA inhibition of nuclear translocation in hepatocytes. The
protein phosphatase inhibitor OA is known to inhibit the PB
induction of CYP2B gene expression in rodent primary hepa-
tocytes (9, 17). Therefore, we examined whether OA repressed
the PB-elicited nuclear localization of CAR. Nuclear extracts
prepared from mouse primary hepatocytes treated with PB for
90 min or pretreated by OA prior to PB induction were sub-
jected to gel shift assays and Western blot analyses. NR1 com-
plex formation in hepatocyte nuclear extracts was markedly
increased by PB induction. Conversely, OA pretreatment pre-
vented the increase in the level of the NR1 complex (Fig. 6).
Western blot analysis unequivocally showed that the nuclear
content of CAR dramatically increased in PB-treated hepato-
cytes, while it was barely detected in nuclear extracts from OA-
plus-PB-treated or nontreated hepatocytes (Fig. 7A). Again,
the nuclear level of CAR in OA-pretreated hepatocytes was as
low as that in nuclear extracts of nontreated hepatocytes. OA
inhibited the PB-induced increase of Cyp2b10 mRNA (Fig.
7B). Thus, OA inhibits the nuclear translocation of CAR in
primary hepatocytes, thus suppressing PB induction of the
General discussion. CAR is a cytoplasmic receptor in liver
and primary hepatocytes which translocates into the nucleus
only after treatment with inducers such as PB and TCPOBOP.
This PB-elicited nuclear translocation is in sharp contrast to an
apparently spontaneous nuclear localization of CAR in trans-
fected HepG2 cells. Recombinant CAR fused with GFP local-
izes to nuclei in transfected HepG2 cells in the absence of the
inducer. 3?-Androstenol does not inhibit the nuclear localiza-
FIG. 1. Transactivation of NR1 by CAR and intracellular localization of GFP-CAR. (A) (NR1)5-tk-luciferase reporter plasmids were cotransfected with GFP-CAR
expression plasmids and pRL-SV40 into HepG2 cells. Sixteen hours later, the transfected cells were treated for another 24 h with 3?-androstenol (Andro.; 4 ?M),
TCPOBOP (250 nM), or both, and luciferase activity was assayed (bottom). To determine intracellular localization, transfected HepG2 cells were induced for 2 h, and
the fluorescence intensity of GFP-CAR was determined by counting 50 HepG2 cells under a microscope and scored as described in the text (top). (B) (NR1)5-tk-
luciferase reporter plasmids were cotransfected with pRL-SV40 into primary hepatocytes. The cells were then treated with 3?-androstenol (4 ?M), TCPOBOP (50 nM),
or both for 24 h, and luciferase activity was assayed.
FIG. 2. Nuclear accumulation of CAR after PB treatment. Forty-microgram
aliquots of total liver nuclear extracts (A) or 30-?l aliquots of the affinity-purified
fractions (B) were used for Western blot analyses. The images were prepared
from 3-min (CAR in panel A), 30-s (RXR? in panel A), and 1-min (CAR in
panel B) exposures. Prestained Protein Marker Broad Range (New England
Biolabs) was used as the molecular mass marker. For the Northern blot (C),
20-?g aliquots of total liver RNAs were electrophoresed, transferred, and hy-
bridized as described in Materials and Methods.
6320 KAWAMOTO ET AL.MOL. CELL. BIOL.
tion of CAR, even at its extremely high concentrations (4 ?M)
compared with known plasma concentrations. Seemingly,
HepG2 cells cannot retain CAR in the cytoplasm, resulting in
constitutive activation of PBREM and expression of the
CYP2B6 gene (19). Since CAR does not require ligand to
transactivate the cis-acting response elements, it is not surpris-
ing to find that CAR is excluded from liver nuclei of non-
treated mice. The nuclear translocation of CAR is tightly reg-
ulated in liver and primary hepatocytes. Consequently, the
constitutively activated CAR can become PB responsive
through a tightly regulated nuclear translocation process. Nu-
clear translocation of CAR is spontaneous in HepG2 cells and
does not require binding by inducers, implying that the CAR
translocation may be ligand independent. This hypothesis of
ligand-independent nuclear localization seems to be supported
by the fact that 3?-androstenol does not affect the nuclear
translocation of CAR in either HepG2 cells or primary hepa-
tocytes. However, it remains to be proven that negative ligands
such as 3?-androstenol and their displacement by PB do not, in
fact, regulate CAR at the step of the nuclear translocation.
The PB-induced CAR nuclear translocation appears to be
regulated by a dephosphorylation-sensitive signaling cascade.
OA treatment inhibits nuclear translocation of CAR as well as
induction of Cyp2b10 mRNA. Glucocorticoid and vitamin D3
receptors are known to undergo ligand-dependent nuclear
translocation in transfected cells (2, 12, 16, 21). These recep-
tors are phosphoproteins (4), and agonist binding is the key
step which initiates a change in the phosphorylation state of
ylates this receptor and decreases its nuclear translocation,
although the phosphorylated receptor remains active in en-
hancing the glucocorticoid enhancer elements. OA also hyper-
phosphorylates the vitamin D3receptor and inhibits its ligand-
FIG. 3. Nuclear accumulation elicited by various PB-type inducers. Liver
nuclear extracts were prepared from mice treated with various xenochemicals.
Total nuclear extracts or NR1 affinity-purified fractions were subjected to West-
ern blot analysis using anti-CAR antibody. Due to different exposure times, band
intensities cannot be accurately compared. In general, exposure times were 3 to
5 min for Western blots of the TCPOBOP and CPZ samples and around 20 min
for Western blots of the o,p?-DDT and PCB samples.
FIG. 4. Cytoplasmic localization of CAR in livers of nontreated mice.
Whole-cell and nuclear extracts were prepared from nontreated (control [C])
and PB-treated (for 3 h) mice as described in Materials and Methods. The
immunoprecipitate was electrophoresed on a sodium dodecyl sulfate–10% poly-
acrylamide gel, transferred, and immunostained by anti-CAR antibody. The
band corresponding to CAR is indicated by an arrow. Intense bands just above
CAR represent heavy-chain IgG. Prestained Protein Marker Broad Range (New
England Biolabs) was used as the molecular mass marker.
FIG. 5. Nuclear localization of CAR after PB treatment. Sections of paraffin-
embedded liver tissue from a PB-treated mouse were incubated with either
anti-CAR antibody (A) or control IgG (C); that from a nontreated mouse was
incubated with anti-CAR antibody (B). Specific immunoreaction of the sections
was visualized colorimetrically by diaminobenzidine.
VOL. 19, 1999 INDUCIBLE NUCLEAR TRANSLOCATION OF THE RECEPTOR CAR6321
dependent nuclear translocation (5). However, the OA Download full-text
concentration affecting glucocorticoid and vitamin D3recep-
tors was 100 nM, 10- to 50-fold higher than the concentrations
inhibiting the nuclear translocation of CAR. Nuclear translo-
cation of CAR appears to be spontaneous in HepG2 cells and
does not require binding of inducer, meaning that the CAR
translocation may be ligand independent. Thus, a cellular
mechanism of the PB-inducible nuclear translocation of CAR
may differ from that regulating the ligand-dependent translo-
cation of glucocorticoid and vitamin D3receptors. Since the
OA-dependent dephosphorylation is essential for the nuclear
translocation of CAR and the induction of CYP2B genes, the
exact mechanism of this signal transduction pathway remains a
question of major interest.
PB displays pleiotropic effects on liver metabolism and phys-
iology, from glucose metabolism to growth regulation and tu-
mor promotion (3, 10, 20). A large number of hepatic genes
are up-regulated by PB, including those enzymes involved in
xenochemical metabolism. Does CAR play a central role in
pleiotropic activation of various genes? The answer to this
question may be yes, although only a few genes, including
CYP2B, CYP3A (19), and the UDPG glucuronosyl transferase
gene UGT1A (13a), are known to be regulated by CAR. CAR
localizes to liver nuclei in response to various PB-type inducers
that transactivate PBREM to induce CYP2B genes, indicating
that nuclear translocation of CAR is a common step in the
regulation of these genes by many, if not all, PB-type inducers.
CAR is now emerging as the major factor mediating a large
number of PB-type inducers and pleiotropically activating nu-
merous genes. Given the fact that CAR is a constitutively
activated receptor, the regulatory mechanism such as an OA-
sensitive nuclear translocation may help explain how CAR
plays the central role in the induction of various genes in
respond to numerous xenochemicals.
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FIG. 6. OA inhibition of PB-dependent increase in the CAR-NR1 complex.
Gel shift assays were performed with the hepatocyte nuclear extracts and32P-
labeled NR1 oligonucleotides. Unlabeled NR1 oligonucleotides (200-fold ex-
cess) were included as the competitor.
FIG. 7. OA inhibition of PB-dependent nuclear localization of CAR. (A)
Western blot analysis was performed with anti-CAR antibody and the nuclear
extracts prepared from nontreated (lane 1), PB-treated (lane 2), and PB-plus-
OA-treated (lane 3) hepatocytes. (B) Total RNAs were subjected to Northern
blot analysis for Cyp2b10 and albumin (Alb.) mRNAs as described in Materials
and Methods. Lanes 1, 2, and 3, RNA samples from control, PB-treated, and
PB-plus-OA-treated hepatocytes, respectively.
6322 KAWAMOTO ET AL.MOL. CELL. BIOL.