Supplement to: Dendritic cell nuclear protein-1, a novel depression-related protein,
up-regulates corticotropin-releasing hormone expression
Tian Zhou,1,
2
Shanshan Wang,3 Haigang Ren,1 Xinrui Qi,1,
2
Sabina Luchetti,2 Willem
Kamphuis,2 Jiang-Ning Zhou,1 Guang-Hui Wang1 and Dick F. Swaab2
Methods and Materials
Specificity of antibody
Neuro2a cells were transfected with enhanced green fluorescent protein (EGFP), an
EGFP-tagged dendritic cell nuclear protein-1 (DCNP1) or DCNP11-116-EGFP construct
separately. After 24h, cells were washed with phosphate buffered saline (pH7.4) and fixed by
4% paraformaldehyde at room temperature. Following permeabilization with 0.25% Triton
X-100 in phosphate buffered saline and blocking with 4% fetal bovine serum, the cells were
incubated with
our
DCNP1-antibody (1:1000), followed
by incubation with a
rhodamine-conjugated donkey anti-rabbit IgG (1:200) (Santa Cruz biotech). Finally, the cells
were studied using an inverted IX71 microscope system (Olympus) (see Fig S1a).
Immunoblot analysis was performed after transfection with plasmids expressing
DCNP1-EGFP, DCNP11-116-EGFP or EGFP alone in human embryonic kidney 293 cells. The
cells were collected 48h after transfection. Proteins were separated by 12% SDS-PAGE and
then
transferred
onto
a
polyvinylidene
difluoride
membrane
(Millipore).
Our
DCNP1-antibody (1:1000), anti- green fluorescent protein (GFP) antibody (Santa Cruz
Biotechnology) (1:1000) and un-immunized rabbit serum were used separately as primary
antibodies for overnight incubation, followed by incubation with sheep anti-rabbit
IgG-Horseradish Peroxidase antibody (1:10000) (Amersham Pharmacia Biotech) as the
secondary antibody. The proteins were visualized using an ECL detection kit (Amersham
Pharmacia Biotech) (see Fig S1b).
Comparison of two anti-DCNP1 antibodies
After deparaffinization and hydration of alternating brain sections, antigen retrieval was
performed in Tris-HCl buffer (pH 9.0) for 10 min at 90 °C, followed by incubation with either
of the two DCNP1-antibodies: one was kindly donated by Masuda, (Masuda et al., 2002) and
another was our polyclonal DCNP1-antibody (1:300). Each alternating section was incubated
for 1h at room temperature and subsequently at 4 °C overnight. The next day, sections were
incubated
with
secondary
horseradish
peroxidase-conjugated
antibody
(1:100)
(Dakocytomation) for 1h at room temperature. Subsequently, sections were incubated for 10
min in tris-buffered saline containing 0.05% 3, 3'-diaminobenzidine (Sigma Chemicals),
0.01% hydrogen peroxide and 0.3% nickel–ammonium sulfate. After being developed,
dehydrated and cleared, the sections were cover-slipped with entellan mounting medium
(Merck) (see Fig S2).
α-GFP
α-DCNP1
A
B
C
D
E
F
G
H
I
J
K
L
DCNP1-EGFP
DCNP11~116-EGFP
EGFP
IgG
DCNP11~116-EGFP
EGFP
DAPI
DCNP1-EGFP
EGFP
DCNP11~116-EGFP
DCNP1-EGFP
α-DCNP1
DCNP1-EGFP
EGFP
DCNP11~116-EGFP
DCNP1-EGFP
EGFP
DCNP11-116-EGFP
RESULTS
Figure S1
a):
MERGE
b):
Figure S1: Specificity of our DCNP1-antibody. (a): Neuro2a cells were transfected with
plasmids expressing EGFP (A-D) as a control, or DCNP1-EGFP (E-H) and DCNP11-116-EGFP
(I-L). Then the cells were incubated with DCNP1-antibody (α-DCNP1) as the primary
antibody, and visualized using a fluorescent microscope (magnification ×400). There was no
staining present in the cells that were transfected with EGFP as a negative control (B) or cells
that did not express DCNP1-EGFP or DCNP11-116-EGFP (indicated by arrowheads, E, F, I
and J). A specific red staining of DCNP1 is seen when the cells were transfected with
DCNP1-EGFP or DCNP11-116-EGFP and stained with our DCNP1-antibody (indicated by
arrow, F and J). The nuclei were stained with blue Hoechst 33342 (C, G and K). The pictures
(D), (H) or (L) represented the merged pictures (A-C), (E-G) or (I-K) which displays that the
cells were located in the same position. This experiment showed that our DCNP1-antibody is
specifically staining DCNP1 but not EGFP. Scale bars: 10μm.
(b): human embryonic kidney 293 cells were transfected with plasmids expressing
DCNP1-EGFP, DCNP11-116-EGFP or EGFP. 48h after transfection, the cells were collected
and
subjected
to
immunoblot
analysis
using
anti-GFP
antibody
(α-GFP),
our
DCNP1-antibody (α-DCNP1) or un-immunized rabbit serum which was as a negative control.
DCNP1-EGFP and DCNP11-116-EGFP can be detected with both anti-GFP antibody and
anti-DCNP1 antibody but not with un-immunized rabbit serum. This test demonstrates that
our DCNP1-antibody stains DCNP1 but not EGFP. KD = kilodalton.
Figure S2
Figure S2: Comparison of the two DCNP1-antibodies. The same numbered cells are stained
in adjacent paraffin sections of a control patient by our DCNP1-antibody (left) and the
DCNP1-antibody from Masuda et al.,2002 (right). There is a clear neuronal staining in both,
the paraventricular nucleus (A, B) and supraoptic nucleus (C, D). By both antibodies the cells
in the supraoptic nucleus stained stronger than in the paraventricular nucleus (which is in
agreement with the quantitative polymerase chain reaction data) (see manuscript Fig 3).
DCNP1-immunostaining was detected in various brain areas in neurons and glia cells, mostly
in the cytoplasm but also in the nucleus (see also manuscript Fig. 2).
Reference for Supplementary Data
Masuda M, Senju S, Fujii Si S, Terasaki Y, Takeya M, Hashimoto Si S, et al. Identification
and immunocytochemical analysis of DCNP1, a dendritic cell-associated nuclear protein.
Biochem Biophys Res Commun 2002; 290: 1022-9.