Departments of
* Biochemistry and
Medicine, Division of Hematology, and
Division of Pathology, Juntendo University, School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
Correspondence: Isao Nagaoka, Department of Biochemistry, Juntendo University, School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. E-mail: nagaokai{at}med.juntendo.ac.jp
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Key Words: superoxide • cytochrome b558 • cytosolic factor
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The importance of each of these oxidase components is demonstrated in chronic granulomatous disease (CGD), which is an inherited disease characterized by mutations that result in the loss or inactivation of one of the core subunits of NADPH oxidase, with the failure of O2- production and a marked increase in the susceptibility of affected patients to bacterial and fungal infections [9 , 12 ]. Previous studies suggested that activity of the oxidase was not expressed in immature neutrophil precursors using bone-marrow cells from patients with no clinical evidence of neutrophil-functional disorders [13 ]. Furthermore, it is known that following maturation of HL-60 cells to neutrophils, superoxide-generating activity is obtained [14 ], and NADPH oxidase components are expressed [15 ]. However, it is not clear at which stage NADPH oxidase activity is acquired during maturation of neutrophil precursors. To clarify the stage(s) of respiratory-burst enzyme expression during neutrophil maturation in this study, we analyzed the expression of NADPH oxidase components using the human promyelocytic leukemia HL-60 cell line as a model system.
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Antibodies
Rabbit anti-gp91phox and anti-p22phox
polyclonal antibodies were prepared using synthetic peptides
(corresponding to CISNSESGPRGVHFIFNKENF and CAGGPPGGPQVNP IPVTDEVV,
respectively), as described previously [17
]. Mouse
anti-gp91phox (7D5) monoclonal antibody (mAb) was kindly
provided by Dr. Michio Nakamura (Nagasaki University, Japan)
[18
]. Rabbit anti-p40phox polyclonal
antibody is raised by using synthetic peptide (MAVAQQLRAESDFEQ), as
described previously [19
]. Mouse
anti-p67phox and anti-p47phox mAbs, provided by
Dr. Hiroyuki Nunoi (Kumamoto University, Japan), were also used
[20
]. Rabbit anti-rac-2 polyclonal antibody was
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse
anti-CD11b mAb D12 was purchased from Becton-Dickinson (San José,
CA).
Cell culture
HL-60 cells were grown in RPMI 1640 medium (Nissui
Pharmaceutical Co. Ltd., Tokyo, Japan) containing 10% fetal bovine
serum, 2 mM L-glutamine, 100 U/ml penicillin, and 0.1 mg/ml
streptomycin at 37°C in a 5% CO2 atmosphere
[21
]. Cell number and viability were determined by
trypan blue exclusion. HL-60 cells (2.5–3.5x105 cells/ml)
were maturated to neutrophils by incubation with 1.3% DMSO for 7 days.
Control HL-60 cells were cultured for 7 days without DMSO.
Preparation of cells
HL-60 cells were collected at 0, 1, 3, 5, and 7 days, and washed
with phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 8.1 mM
Na2HPO4, 1.5 mM KH2PO4,
pH 7.4). For Western blot analysis, the cells were treated with 5 mM
diisopropyl fluorophosphate for 30 min at 4°C and washed in PBS. The
cells were finally suspended in PBS at 108/ml and stored at
-80°C. Human bone-marrow cells were obtained by aspiration from the
superior iliac crest of healthy volunteers. Bone-marrow cells were
immediately anticoagulated in heparin and diluted in an equal volume of
PBS. After sedimentation of erythrocytes in 1% dextran and hypotonic
lysis of residual erythrocytes, leukocyte-rich cells were obtained.
Cytocentrifuge preparations were made using Cytospin 2 (105
cells/slide; 340 rpm, 5 min; Shandon Instruments, Pittsburgh,
PA), stained with May/Grünwald/Giemsa and examined by
light microscopy. Each morphological subtype of neutrophil lineage
cells was identified based on conventional criteria (cell size, ratio
of nucleus to cytoplasm, and characteristics of nuclear chromatin). In
addition, CD11b was used as a myeloid maturation marker in cytochemical
analysis, as described below. Based on these criteria when at least 500
cells were scored, leukocyte-rich preparations of human bone-marrow
cells proved to contain approximately 5% eosinophilic cells, 11%
mononuclear cells (
9% lymphocytes and 2% monocytes), and 84%
neutrophil lineage cells (4% promyelocytes, 15% myelocytes,
21% metamyetocytes, 26% band cells, and 18% segmented
cells).
Assay of NADPH oxidase activity
During maturation, acquisition of
O2--generating activity was determined by
nitro blue tetrazolium (NBT) assay, which detects reduction of NBT to
formazan by superoxide oxide. The assay was performed based on the
method of Zakhireh and Root [13
] with a slight
modification by incubating cells (1x106 cells/ml, 200
µl) with 0.04% NBT in PBS containing 1 mM CaCl2 and 1 mM
MgCl2 in the presence or absence of 500 ng/ml PMA at 37°C
for 30 min in a Lab-Tak chamber slide (Nalge-Nunc International,
Naperville, IL), and then formazan-containing cells were
monitored. Furthermore, NADPH oxidase activity was assayed by
cytochrome c reduction [20
]; the assay mixtures
consisted of 1 x 106 cells/ml, 60 µM cytochrome c,
1 mM CaCl2, 1 mM MgCl2, and 1 µg/ml PMA with
or without 20 µg/ml superoxide dismutase in a total volume of 400
µl PBS. After stimulation at 37°C for 5 min, the mixtures were
centrifuged at 800 g for 5 min. Cytochrome c reduction was
calculated by the absorbance difference at 540–550 nm using an
absorption coefficient of 21,000 M-1
cm-1.
Isolation of RNA and Northern blot analysis
Total cellular RNA was isolated from HL-60 cells by the acid
guanidinium thiocyanate-phenol-chloroform extraction method
[22
]. RNA (2.5 µg) was separated by electrophoresis on
1.2% agarose-formaldehyde gel and transferred by capillary blotting
onto nylon membranes (Hybond N+, Amersharm-Pharmacia
Biotech., Bukinghamshire, UK). RNA was crosslinked with a Funa-UV
Linker (Funakoshi Co. Ltd., Tokyo, Japan), and the blots were
hybridized with cDNA probes, which were labeled with digoxigenine-high
prime DNA labeling kit (Roche Diagnostics, Mannheim, Germany). The
0.45-kb gp91phox cDNA (encompassing nt 800–1246)
[23
], the 0.47-kb p22phox cDNA (encompassing
nt 200–666) [24
], the 0.57-kb p67phox cDNA
(encompassing nt 555–1128) [25
], the 0.47-kb
p47phox cDNA (encompassing nt 354–826)
[26
], the 0.54-kb p40phox cDNA (encompassing
nt 107–645) [27
], and the 0.45-kb rac-2 cDNA
(encompassing nt 72–517) [28
] were obtained by the
amplification of human bone-marrow cell cDNA with polymerase chain
reaction. The 2.3-kb 
-actin cDNA was graciously provided by P.
Gunning and L. Kedes (Stanford University, CA). To measure the relative
amounts of mRNA, the detected bands were quantified using a scanning
densitometer (MasterScan System, Scanalytics, Inc., Fairfax, VA).
Western blot analysis
Western blot analysis was performed as described previously
[20
]. The amounts of protein in each sample were
quantitated with a Pierce BCA protein assay kit (Pierce Chemical Co.,
Rockford, IL), according to the manufacturer’s instruction. HL-60
cells were solubilized in sample buffer (62.5 mM Tris-HCl pH 6.8, 2%
sodium dodecyl sulfate, 10% glycerol, 0.005% bromophenol blue, 5%
ß-mercaptoethanol), disrupted in ice by sonication (Tomy Ultrasonic
Disruptor UD-201, Tominaga Works Ltd., Tokyo, Japan), and denatured at
100°C for 2 min. Then, the samples (10 or 20 µg protein) were
subjected to 12% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE). The separated proteins were
electrophoretically transferred to nitrocellulose membranes (Schleicher
& Schuell, Dassel, Germany), and the membranes were blocked in Block
Ace (Dainippon Pharmaceutical Co.) and probed with rabbit
anti-gp91phox (1:1000), anti-p22phox (1:2000),
anti-p40phox (1:1000), and anti-rac-2 (0.5 µg/ml)
polyclonal antibodies or mouse anti-p67phox (1:5000) and
anti-p47phox (1:5000) mAbs. After washing, the membranes
were further probed with a 1:5000 dilution of horseradish
peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG) (ICN
Pharmaceuticals, Inc., Costa Mesa, CA) or goat anti-mouse IgG/IgM
(Kirkegaard and Perry Laboratories, Gaithersburg, MD). The oxidase
components were finally detected with an ECL Western blotting detection
system (Amersham-Pharmacia Biotech.). The detected bands were
quantified using the MasterScan System (Scanalytics).
Double immunofluorescence staining
Cytocentrifuge preparations were fixed in 10% formaldehyde/PBS
for 10 min and washed in PBS-0.05% Tween 20. For staining with
anti-CD11b mAb, cells were fixed in acetone for 5 min. Then, slides
were blocked with 2% normal goat serum for 20 min and incubated with
rabbit anti-p22phox (1:500), anti-rac-2 (2 µg/ml), or
anti-p40phox (1:250) polyclonal antibody overnight at 4°C
in a moist chamber. After washing, the slides were incubated with a
1:300 dilution of biotin-conjugated goat anti-rabbit IgG (DAKO A/S,
Glostrup, Denmark) for 60 min at room temperature and further incubated
with fluorescein isothiocyanate (FITC)-conjugated streptavidine (DAKO)
(1:100) for 30 min in the dark. For double staining, the slides were
blocked with the avidin-biotin blocking kit (Vector Laboratories,
Burlingame, CA). Then, the slides were incubated with mouse
anti-gp91phox (7D5, 5 µg/ml), anti-p67phox (3
µg/ml), anti-p47phox (3 µg/ml), or anti-CD11b (D12, 10
µg/ml) mAb overnight at 4°C. After washing, the slides were
incubated with a 1:300 dilution of biotin-conjugated goat anti-mouse
IgG (DAKO) for 60 min at room temperature and further incubated with
Alexa 594-conjugated streptavidine (Molecular Probes, Inc., Eugene, OR)
(1:100) for 30 min. After washing, the slides were observed with a
Ziess Axiophot photomicroscope (Carl Zeiss, Inc., Oberkochen, Germany).
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 View larger version (18K): [in a new window] |
Figure 1. Maturation of HL-60 cells to neutrophil lineage by treatment with DMSO.
(A) HL-60 cells were cultured in the presence of 1.3% DMSO for 7 days.
On 1, 3, 5, or 7 days of culture, cells were collected and stained with
May/Grünwald/Giemsa. More than 200 cells were counted, and the
percentage of each subtype of neutrophil lineage cells ( ,
promyelocyte;  , myelocyte;  , metamyelocyte; •, band cell;  ,
segmented cell) was calculated. Data represent the mean ±
SD of three separate experiments. (B) Evaluation of
superoxide-generating activity of DMSO-induced HL-60 cells. After
culture with () or without (•) 1.3% DMSO for 1, 3, 5, and 7 days,
HL-60 cells (1x106 cells/ml) were incubated with 0.04%
NBT and 500 ng/ml PMA at 37°C for 30 min. More than 200 cells were
counted under microscope, and the percentage of formazan-containing
cells was calculated. Data represent the mean ± SD of
three separate experiments.
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Evaluation of the expression of NADPH oxidase components in
maturating HL-60 cells
We evaluated the expression of mRNAs and proteins for NADPH
oxidase components in maturating HL-60 cells by Northern blot and
Western blot analyses, respectively. First, we examined the expression
of mRNAs for gp91phox, p22phox,
p67phox, p47phox, p40phox, and
rac-2. As shown in Figure 2
, gp91phox and p67phox mRNAs were not
detectable in uninduced and 1-day-induced HL-60 cells, whereas
p47phox was detected on day 1 of induction. Thereafter,
these mRNAs were increased with maturation. In contrast, small amounts
of p22phox, p40phox, and rac-2 mRNAs were
constitutively expressed in uninduced HL-60 cells and increased with
maturation. When RNA samples were analyzed with -actin cDNA probe,
almost the same amounts of -actin mRNA transcripts were detected in
all RNA preparations.
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Figure 2. Northern blot analysis of mRNAs for NADPH oxidase components. (A)
Northern blot analysis. Total cellular RNA (2.5 µg) was
electrophoresed on an agarose/formaldehyde gel and blotted onto a nylon
membrane. The blot was hybridized with DIG-labeled
gp91phox, p22phox, p67phox,
p47phox, p40phox, rac-2, or -actin cDNA
probe. Shown are the results with uninduced HL-60 cells (0 day) and
HL-60 cells incubated with 1.3% DMSO for 1, 3, 5, or 7 days. In
addition to a 1.2-kb p40phox mRNA, a 1.5-kb splice variant,
p40phox mRNA, is expressed in human myeloid cells
[27
]. A 2.2-kb fainter band is consistently observed
above a 1.5-kb intense band of p47phox mRNA using RNA of
induced HL-60 cells [29
]. (B) Relative amounts of
gp91phox, p22phox, p67phox,
p47phox, p40phox, rac-2, and -actin mRNAs.
mRNA bands detected were quantified using the MasterScan system. Each
mRNA level is expressed as a ratio to the maximum mRNA level. Data
represent the mean ± SD of three separate
experiments.
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Figure 3. Western blot analysis of proteins for NADPH oxidase components. (A)
HL-60 cell sonicates (10 µg protein for p22phox,
p67phox, p47phox, p40phox, and
rac-2; 20 µg protein for gp91phox) were subjected to
SDS-PAGE, and proteins were detected by immunoblot analysis using
anti-gp91phox, anti-p22phox,
anti-p67phox, anti-p47phox,
anti-p40phox, or anti-rac-2 antibody. The results are shown
with uninduced HL-60 cells (0 day) and HL-60 cells incubated with 1.3%
DMSO for 1, 3, 5, or 7 days. (B) Relative amounts of
gp91phox, p22phox, p67phox,
p47phox, p40phox, and rac-2. Protein bands
detected were quantified using the MasterScan system. Each protein
level is expressed as a ratio to the maximum protein level. Data
represent the mean ± SD of three separate
experiments.
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Figure 4. Evaluation of the expression of NADPH oxidase components in
DMSO-treated HL-60 cells by double immunocytochemical staining. HL-60
cells were incubated with 1.3% DMSO for 5 days. After fixation, the
cells were incubated with rabbit anti-p22phox (E and H),
anti-rac-2 (F), or anti-p40phox (G) antibody and then
stained with biotin-conjugated goat anti-rabbit IgG and FITC-conjugated
streptavidine. After blocking, the cells were further incubated with
mouse anti-gp91phox (A), anti-p67phox (B),
anti-p47phox (C), or anti-CD11b (D) mAb and then stained
with biotin-conjugated goat anti-mouse IgG and Alexa 594-conjugated
streptavidine. Phase photomicrographs of the same field are shown in
panels I (A/E), J (B/F), K (C/G), and L (D/H), repectively. More than
200 cells were evaluated in each slide. Promyelocytes (PM) were
negative for gp91phox, p67phox,
p47phox, and CD11b (A–D) but positive for
p22phox, rac-2, and p40phox (E–H). All oxidase
components were strongly positive in myelocytes (MC), metamyelocytes
(MM), band cells (Band), and segmented cells (Seg). Original
magnification x 600.
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Figure 5. Evaluation of the expression of NADPH oxidase components in human
bone-marrow cells by double immunocytochemical staining. After
fixation, human bone-marrow cells were incubated with rabbit
anti-p22phox (E and H), anti-rac-2 (F), or
anti-p40phox (G) antibody and then stained with
biotin-conjugated goat anti-rabbit IgG and FITC-conjugated
streptavidine. After blocking, the cells were further incubated with
mouse anti-gp91phox (A), anti-p67phox (B),
anti-p47phox (C), or anti-CD11b (D) mAb and then stained
with biotin-conjugated goat anti-mouse IgG and Alexa 594-conjugated
streptavidine. Phase photomicrographs of the same field are shown in
panels I (A/E), J (B/F), K (C/G), and L (D/H), respectively. More than
200 cells were evaluated in each slide. gp91phox,
p67phox, p47phox, p22phox, and
CD11b were detected in myelocytes (MC), metamyelocytes (MM), band cells
(Band), and segmented cells (Seg) (A–E and H), whereas rac-2 and
p40phox were detected from the promyelocyte stage (PM) (F
and G). Original magnification x 600.
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Finally, cellular stages of superoxide-producing cells were evaluated
using DMSO-treated HL-60 cells by NBT assay. By stimulation with PMA,
cells were stained at the stages of myelocyte, metamyelocyte, band
cell, and segmented cell; however, promyelocytes were not stained
(Fig. 6B
). Almost the same results were obtained by NBT assay using human
bone-marrow cells; cells after myelocyte stages were stained by NBT
(Fig. 6C)
. It is known that eosinophils and monocytes/macrophages also
express NADPH components and produce superoxide [5
,
6
, 20
]. In the bone-marrow cell
preparations, eosinophilic cells and monocytes were <10%, and
neutrophil lineage cells (promyelocytes, myelocytes, metamyetocytes,
band cells, and segmented cells) were 84%. Furthermore, >80% of
cells were positive for immunocytochemical staining (NADPH oxidase
components) and NBT assay (superoxide production) among
bone-marrow-derived, leukocyte-rich cells (unpublished results). Thus,
positive cells for immunocytochemical staining and NBT assay likely
represent the neutrophil lineage cells in the bone marrow.
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Figure 6. Evaluation of the superoxide-generating activity by NBT assay. HL-60
cells were induced with 1.3% DMSO for 5 days and incubated with 0.04%
NBT in the absence (A) or presence (B) of 500 ng/ml PMA at 37°C for
30 min. Human bone-marrow cells were also incubated with 0.04% NBT in
the presence of 500 ng/ml PMA (C). Only myelocyte (MC), metamyelocyte
(MM), band cell (Band), and segmented cell (Seg) are positive in B and
C. All cells are negative in A. Original magnification x 500.
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rac-1 and rac-2 are low molecular-weight guanosine 5'-triphosphate (GTP)-binding proteins involved in the activation of NADPH oxidase [9 10 11 ]. rac-2, 92% homologous with rac-1 [28 ], is present predominantly in human neutrophils, and >96% of rac proteins are rac-2 in human neutrophil [37 ]. Northern blot and Western blot analyses have revealed that rac-2 is expressed in uninduced HL-60 cells and increased during neutrophil maturation. Furthermore, rac-2 could be detected at the promyelocyte stage by immunocytochemical staining. These observations indicate that rac-2 is constitutively expressed in uninduced HL-60 cells and increased following neutrophil maturation.
It has been reported that NADPH oxidase activation is fully restored by cytochrome b558, p67phox, p47phox, and rac proteins [38 , 39 ]. In this study, we have shown that p67phox is expressed later than p47phox, during neutrophil maturation at mRNA and protein levels. This observation is consistent with the concept that p67phox is the limiting cytosolic component required for the expression of cytosol oxidase activity during maturation of HL-60 cells [15 , 36 ].
p40phox is originally identified as the fourth cytosolic component in guinea pig and human neutrophils by us and other investigators [40 41 42 ]. However, it has not been clear whether p40phox is an essential oxidase component. It is interesting that we have shown that the synthetic peptide corresponding to the amino acid sequence of p40phox inhibits the translocation of cytosolic components and NADPH oxidase activation [43 ]. In addition, an antibody to the p40phox C-terminus suppresses NADPH oxidase activation [19 ]. These observations suggest that p40phox likely regulates activation of the oxidase. In contrast, it is reported using cell-free reconstitution and whole-cell cotransfection techniques that p40phox is involved in the down-regulation of NADPH oxidase activity [44 ]. Thus, p40phox is assumed to have a role in modulating the activity of NADPH oxidase. The present results, that expression of p40phox and superoxide-producing activity is increased in parallel with neutrophil maturation, also suggest that p40phox may be involved in the activation of NADPH oxidase.
The observations with Western blot and Northern blot analyses were supported by the results of immunocytochemical staining, where gp91phox, p67phox, and p47phox were detected after myelocyte stages, but p22phox, p40phox, and rac-2 were detected from the promyelocyte stage of HL-60 cells. Furthermore, NBT assay has indicated that superoxide can be produced after the myelocyte stage but not before the promyelocyte stage, although some components of NADPH oxidase (p22phox, p40phox, and rac-2) are expressed at the promyelocyte stage of HL-60 cells. In addition, using human bone-marrow cells, we have obtained almost the same results as those with HL-60 cells by immunocytochemical staining and NBT assay. However, p22phox could not be detected in promyelocytes of human bone-marrow cells by immunocytochemical staining. In HL-60 cells, neither gp91phox mRNA nor its protein was expressed, whereas p22phox mRNA and protein were detected at the promyelocyte stage. This is unusual, because gp91phox and p22phox are usually missing in A-22 CGD and in X-91 CGD, and these subunits are believed to stabilize each other [45 ]. Actually, gp91phox and p22phox could be detected after myelocyte stages in human bone-marrow cells. However, it has been reported recently that only p22phox, not p91phox, is expressed in vascular smooth muscle cells [46 ] and that gp91phox and p22phox can be expressed separately in transgenic COS7 cells [47 ]. Thus, the difference in the expression of p22phox we observed may be based on the characteristics of cells used (normal human bone-marrow cells vs. promyelocytic leukemia cell line HL-60).
Transcription factors involved in the expression of gp91phox, p47phox, p40phox, and rac-2 have been investigated. SP1/3 are reported to be important for murine rac-2 promoter activity [48 ], and AP-2 and NF-E1 are assumed to be involved in the expression of p40phox [49 ]. In contrast, PU.1 is known to be essential for the expression of gp91phox and p47phox [50 51 52 ]. Moreover, the expression of PU.1 increases gradually to the myelocytic stage during differentiation/maturation of myeloid cells [32 , 53 ]. Consistent with these observations, the present study has revealed that gp91phox and p47phox can be expressed at the myelocyte stage in DMSO-treated HL-60 cells and human bone-marrow cells.
In contrast with our observations, it was previously shown, using the NBT assay, that the activity of NADPH oxidase was not expressed until the metamyelocyte stage using bone-marrow cells from individuals with disorders, such as miliary tuberculosis, idiopathic thrombocytopenic purpura, chronic renal failure, and multiple myeloma [13 ]. The discrepancy between the previous results and our results may be because of the difference in the cells used (from individuals with disorders vs. healthy volunteers) and/or assay conditions for NBT assay (0.2% NBT and 100 ng/ml PMA vs. 0.04% NBT and 500 ng/ml PMA).
In summary, the present study indicates that all the components for NADPH oxidase are expressed, and the superoxide-producing activity is obtained after myelocyte stages during neutrophil maturation.
Received November 9, 1999; revised April 13, 2000; accepted April 17, 2000.
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activates a p22phox-based NADPH oxidase in vascular smooth muscle Biochem. J. 329,653-657This article has been cited by other articles:
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S.-L. Li, A. J. Valente, M. Qiang, W. Schlegel, M. Gamez, and R. A. Clark Multiple PU.1 sites cooperate in the regulation of p40phox transcription during granulocytic differentiation of myeloid cells Blood, May 29, 2002; 99(12): 4578 - 4587. [Abstract] [Full Text] [PDF]
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J.-M. Li and A. M. Shah Intracellular Localization and Preassembly of the NADPH Oxidase Complex in Cultured Endothelial Cells J. Biol. Chem., May 24, 2002; 277(22): 19952 - 19960. [Abstract] [Full Text] [PDF]
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M. Chi, S. Tridandapani, W. Zhong, K. M. Coggeshall, and R. F. Mortensen C-Reactive Protein Induces Signaling Through Fc{gamma}RIIa on HL-60 Granulocytes J. Immunol., February 1, 2002; 168(3): 1413 - 1418. [Abstract] [Full Text] [PDF]
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K. A. Gauss, P. L. Bunger, and M. T. Quinn AP-1 is essential for p67phox promoter activity J. Leukoc. Biol., January 1, 2002; 71(1): 163 - 172. [Abstract] [Full Text] [PDF]
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S.-L. Li, A. J. Valente, L. Wang, M. J. Gamez, and R. A. Clark Transcriptional Regulation of the p67phox Gene. ROLE OF AP-1 IN CONCERT WITH MYELOID-SPECIFIC TRANSCRIPTION FACTORS J. Biol. Chem., October 12, 2001; 276(42): 39368 - 39378. [Abstract] [Full Text] [PDF]
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T. Hasebe, J. Hua, A. Someya, P. Morain, F. Checler, and I. Nagaoka Involvement of cytosolic prolyl endopeptidase in degradation of p40-phox splice variant protein in myeloid cells J. Leukoc. Biol., June 1, 2001; 69(6): 963 - 968. [Abstract] [Full Text] [PDF]
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