Supplementary materials and methods and Tables
Supplementary materials and methods
Antibodies
For western blot - A polyclonal rabbit anti-p33MONOX antibody was raised against the
sequence “NPSTMDSGSGDKDR” (aa 204-217 of p33MONOX) from BioGenes GmbH (Berlin,
Germany). Antibody specificity was tested using recombinantly expressed p33MONOX in
E.coli, yeast and in mammalian cells as well as by ELISA (not shown) and used at 1:10000
dilution. The following primary antibodies were also used: anti-beta-actin (Actb, 1:3000, goat
polyclonal; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-App (1:10000, rabbit
polyclonal; Sigma-Aldrich, WI, USA), anti-phosphorylated App (pApp) (1:1000, rabbit
polyclonal; Cell Signaling Technology Inc., Danvers, MA, USA), anti-Bcl2 (1:2000, rabbit
polyclonal; Cell Signaling), anti-pBcl2 (Ser70, 1:500, rabbit polyclonal; Santa Cruz), anti-Erk1/2
(extracellular-signal-regulated kinases 1/2, also known as mitogen-activated protein kinase 1/3
(Mapk1/3), 1:500, rabbit polyclonal; Chemicon, Temecula, CA, USA), anti-pErk1/2
(phosphorylated Erk1/2, 1:2000, rabbit polyclonal; Santa Cruz), anti-Gapdh (G1yceraldehyde-3phosphate dehydrogenase, 1:10000, mouse monoclonal; Santa Cruz). The following secondary
antibodies were used for western blotting purposes: anti-rabbit (1:5000, goat polyclonal; GE
Healthcare UK Limited, Buckinghamshire, UK) and anti-mouse (1:5000, goat polyclonal; GE
Healthcare). The mouse anti-Cobra1 antibody was kindly provided by Prof. Rong Li (The
University of Texas Health Science Center at San Antonio, Department of Molecular Medicine /
Institute of Biotechnology, 15355 Lambda Drive, San Antonio, TX, 78245-3207, USA) (1) and
we also purchased mouse, monoclonal anti-Cobra1 antibody from Abnova (Taiwan).
For immunocytochemistry (ICC) and Immunohistochemistry (IHC) – The following primary
antibodies were used: anti-p33MONOX (1:500), anti-Mtap2 (microtubule-associated protein 2,
1:500, mouse monoclonal; Chemicon), and anti-Syp (synaptophysin, 1:500, mouse monoclonal;
Chemicon), anti-Tuba1a (Tubulin alpha, 1:500, mouse monoclonal; Santa Cruz). The following
secondary antibodies labeled with Fluorescein isothiocyanate (FITC) were used: Alexa Fluor®
488 anti-rabbit (1:400, goat polyclonal; Molecular Probes, Eugene, OR, USA) and Alexa Fluor®
568 anti-mouse (1:400, goat monoclonal; Molecular Probes).
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Cell culture
Rat B104 neuroblastoma, human SHSY5Y neuroblastoma, rat PC12 (rat adrenal
pheochromocytoma) and HEK293T (human embryonic kidney) cells (American Type Culture
Collection (ATCC, Manassas, VA, USA)) were maintained in complete media containing
Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Invitrogen, Carlsbad, CA, USA), 10 %
fetal bovine serum (FBS; Gibco), and 1 % 100 μg/ml penicillin/streptomycin (Gibco). All cells
were cultured at 37 °C in humidified 5 % CO2/ 95 % air incubator unless otherwise stated (2-5).
PC12 cells were differentiated with nerve growth factor (Ngf, 100 ng/ml; Invitrogen) for 10
days.
Stable transfection of SHSY5Y, B104 and PC12 cells
Using the ViraPower™ Lentiviral Expression Systems (Invitrogen) protocol, HEK293T cells
were maintained and transfected with a pLenti expression vector containing the gene of interest
(p33 in EF.CMV.GFP-Lentivector (ATCC)). After 12 h, Lipofectamine™ 2000 (Invitrogen)containing media was aspirated and fresh complete media was added. The virus was collected
from the cell culture supernatant by centrifugation (4000xg/ 15 min/ 4 °C) after 72 h. The
supernatant was directly used to infect SHSY5Y, B104 and PC12 cells with p33 for 36 h. Within
30 h, the green fluorescence (caused by the green fluorescence protein (GFP) co-expressed with
p33) was observed. As control, mock-transfection was performed with a GFP lentivector. A pure
population of p33 expressing cells were obtained through fluorescence-activated cell sorting
(FACS; BD FACSAria Flow Cytometer, BD Biosciences, Singapore) (5).
Transient transfection of B104 cells
A p33Monox-DsRed expression construct was generated by inserting the p33Monox cDNA inframe with the red fluorescent protein DsRed (pDsRed-Express-N1; BD Biosciences Clontech,
Palo Alto, CA, USA) at the C-terminus of p33MONOX (p33-CT-DsRed). B104 cells were
transiently transfected with the p33-CT-DsRed expression vector or empty plasmid (controls, C)
using the Lipofectamine™ 2000 (Invitrogen) transfection reagent (according to the
manufacturer’s protocol). The transfected cells were then visualized by fluorescence microscopy
(Nikon eclipse TE2000U, Nikon, Singapore) (5, 6).
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Animal material
All experimental procedures, including the killing of animals for the purpose of our studies,
were conducted in accordance with International Guiding Principles for Animal Research
(WHO) and permitted by the local Institutional Animal Care & Use Committee (NTU-IACUC).
Mouse tissues were isolated (C57BL/6J mice obtained from the animal facility centre at the
National University of Singapore) after humane killing of the animals using approved anaesthetic
methods (7).
Immunocytochemistry (ICC)
PC12 cells were differentiated in the presence of Ngf (100 ng/ml (Invitrogen) with 10 % FBS
for ten days and then fixed with 4 % paraformaldehyde for 10 min at room temperature (RT).
The cells were washed three times with PBS and blocked in PBS containing 0.01 % Triton-X100 (USB Corporation, Cleveland, OH, USA) and 10 % normal goat serum (Vector
Laboratories, Burlingame, CA, USA) prior to the incubation with the different primary
antibodies overnight at 4 0C. Next day, after three washes in PBS, each sample was incubated for
1 hr in a secondary antibody labelled with FITC (AlexaFluor 568, goat polyclonal anti-rabbit,
1:400, or AlexaFluor 488, goat monoclonal anti-mouse, 1:400; Molecular Probes, Eugene, OR,
USA) for 1 hr. After three washes, the coverslips were mounted onto glass slides using
DAPI/Antifade glue (Chemicon) and analyzed with a Carl Zeiss Live imaging microscope
(Axiovert 200; Carl Zeiss, Göttingen, Germany) (5).
Mouse brain perfusion and Immunehistochemistry (IHC)
Adult mice were anaesthetized by intraperitoneal injection of ketamine/xylazine (RBI, Natick,
MA, USA) and perfused with chilled isotonic saline solution followed by 4 % paraformaldehyde
at RT. Brains were post-fixed for 3 h and protected in a 30 % sucrose solution in 0.1 M sodium
phosphate buffer (PBS) (pH 7.4) at 4 °C for 48-72 hrs. Thereafter, brains were snap frozen in dry
ice cooled isopentane and stored at − 70 °C until sectioning. Brains were sectioned into 20 μm
slices using a Leica cryostat (Leica CM3050S, Leica CM3050S, Leica Microsystems, Nussloch,
Germany). The tissue was washed three times and incubated with PBS containing 0.1 % saponin
and 5 % fetal bovine serum (FBS, Gibco) for 60 min at RT to block non-specific binding and
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then incubated with anti-p33MONOX overnight at 4 °C. This was followed by washes and a 60
min incubation step at RT with a secondary antibody labelled with FITC. Slices were washed
again and then incubated with DAPI 10 g/ml (Sigma) for 1 min followed by five more washes
and then mounted using fluorescence mounting media (Dako, Denmark). Stained slices were
analyzed with a Carl Zeiss Live imaging microscope (Axiovert 200) (5, 7, 8).
P33MONOX protein sequence analyses
P33MONOX cDNA and protein sequences were searched in the National Center for
Biotechnology information (NCBI) Blastp 2.0 program against non redundant GenBank CDS
translations þPDB þSwissProt þPIR þPRF databases, in addition to the UniGene and UniProtKB
databases. Homology search was performed using the Blast and FASTA (Wisconsin Package
Version 10.0, Genetics Computer Group (GCG), Madison, WI) algorithms and hits were aligned
using BestFit (Wisconsin Package Version 10.0, GCG). NetPhos 2.0 protein phosphorylation
prediction server was used to search for phosphorylation sites on p33MONOX. PSORT was
used for predicting cellular localization (5, 6).
SDS-PAGE and western blotting
Protein samples were prepared for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) by
combining 20 µg of cell lysates with Laemmli buffer (187.5 mM Tris-HCl, 30 % glycerol, 15 %
β-mercaptoethanol, 6 % SDS, 0.1 % bromophenol blue, pH 6.8). The samples were boiled for 5
mins and resolved using a (10-12 %) polyacrylamide-SDS gel at 120 Volts (V) at RT. The
resolved proteins were transferred to a polyvinylidene diflouride (PVDF) membrane (0.45 µm;
BioRad Laboratories, Hercules, California, USA) via ‘wet’ transfer method (Biorad) at 55 V, 3 h
at 4 °C. Membranes were blocked for 1 h at RT with 5 % skim milk diluted in PBS with 0.1%
Tween-20 (PBST). Thereafter, membranes were incubated in primary antibody in 5 % milk
overnight at 4 °C followed by washing steps with PBST and incubation in respective secondary
antibodies for 1 h at RT. Subsequently, the Supersignal West Femto Maximum Sensitivity
Substrate (Pierce Biotechnology, Inc., Rockford, IL, USA) was added and the signal was
visualised on a X-ray film (Konica Minolta Inc., Japan) using the Kodak X-OMAT 2000
processor (Kodak, Ontario, Canada) (5, 7, 8).
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Yeast two-hybrid-system screening to identify p33MONOX interacting partners
The two-hybrid-system is an in-vivo yeast-based system that identifies the interaction between
two proteins (for instance X = p33MONOX and Y = human brain cDNA library or COBRA1) by
reconstituting an active transcription factor. The analysis was performed according to the
manufacturer’s protocol (Invitrogen’s brain ProQuest™ two-hybrid-system with Gateway™
technology, Singapore) using p33MONOX as bait. In the ProQuest™ two-hybrid-system, in
comparison to standard two-hybrid-systems, false positives are reduced because three
independent transcription events (from distinct promoters) must occur at independent
chromosomal loci. Positive clones were confirmed by retransformation assays (6). NCBI,
EMBL-EBI (European Bioinformatics Institute) and SIB (Swiss Institute of Bioinformatics)
databases were used to identify the nucleolar protein NOL12 and the prion protein PRNP as
additional p33MONOX interacting proteins (Table-1).
Co-immuneprecipitation (Co-IP)
For immunoprecipitation, p33MONOX-transfected SHSY5Y cells were lysed in lysis buffer
(150 mM Tris-HCl (pH 7.4), 150 mM NaCl, 40 mM NaF, 5 mM EDTA, 1 mM sodium
orthovanadate, 1 % (vol/vol) Nonidet P-40, 0.1 % (wt/vol) sodium deoxycholate, 1 % (vol/vol)
Triton-X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10 ng/ml of aprotinin),
followed by centrifugation at 14,000 rpm at 4 ºC for 15 min. After protein quantification, equal
amounts of the protein supernatants were incubated with polyclonal rabbit p33MONOX
antibody-loaded protein A/G-Sepharose at 4 ºC overnight. As a control, the lysates were
incubated with non-specific rabbit pre-immune serum-loaded protein A/G-Sepharose. The
immunoprecipitates were then washed five times in lysis buffer, and the bound proteins were
recovered by boiling the beads in 2x SDS sample buffer and separated by SDS−PAGE, followed
by western blot with a specific monoclonal mouse anti-COBRA1 antibodya nd the p33MONOX
antibody (6).
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Tables
Table 1: P33MONOX interacts with:
COBRA1
NOL12 (14)
PRNP (16)
Table 2: NOL12 interacts with:
SAP18
CDK4
SF3B3
The Cofactor of BRCA1 is a newly characterized
member of the negative elongation factor (NELF)
complex: involved in gene transcription and splicing
control (9-13).
Nucleolar protein 12, also known as Ribosomal RNA
processing protein 17 or NOP25 (15).
Also known as the major prion protein PrP; exact
function not known. PrP is encoded in the host
genome and is expressed both in normal and infected
cells. Isoform 2 may act as a growth suppressor by
arresting the cell cycle at the G0/G1 phase (17, 18).
Sin3A-associated protein: Histone acetylation plays a
key role in the regulation of eukaryotic gene
expression. Histone acetylation and deacetylation are
catalyzed by multisubunit complexes. The protein
encoded by this gene is a component of the histone
deacetylase complex, which includes SIN3, SAP30,
HDAC1, HDAC2, RbAp46, RbAp48, and other
polypeptides. This protein directly interacts with
SIN3 and enhances SIN3-mediated transcriptional
repression when tethered to the promoter (19, 20).
Cyclin-dependent kinase 4: the protein encoded by
this gene is a member of the Ser/Thr protein kinase
family. This protein is highly similar to the gene
products of S. cerevisiae cdc28 and S. pombe cdc2. It
is a catalytic subunit of the protein kinase complex
that is important for cell cycle G1 phase progression.
The activity of this kinase is restricted to the G1-S
phase, which is controlled by the regulatory subunits
D-type cyclins and the CDK inhibitor p16 (INK4a).
This kinase was shown to be responsible for the
phosphorylation of retinoblastoma gene product (Rb).
Mutations in this gene as well as in its related
proteins including D-type cyclins, p16 (INK4a) and
Rb were all found to be associated with
tumorigenesis of a variety of cancers (21).
Splicing factor 3b, subunit 3: this gene encodes
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SOD2
SLC25A38
subunit 3 of the splicing factor 3b protein complex.
Splicing factor 3b, together with splicing factor 3a
and a 12S RNA unit, forms the U2 small nuclear
ribonucleoproteins complex (U2 snRNP). The
splicing factor 3b/3a complex binds pre-mRNA
upstream of the intron's branch site in a sequence
independent manner and may anchor the U2 snRNP
to the pre-mRNA. Splicing factor 3b is also a
component of the minor U12-type spliceosome.
Subunit 3 has also been identified as a component of
the STAGA (SPT3-TAF(II)31-GCN5L acetylase)
transcription
coactivator-HAT
(histone
acetyltransferase) complex, and the TFTC (TATAbinding-protein-free TAF(II)-containing complex).
These complexes may function in chromatin
modification, transcription, splicing, and DNA repair
(22).
Superoxide dismutase 2: this gene is a member of the
iron/manganese superoxide dismutase family. It
encodes a protein that forms a homotetramer and
binds one manganese ion per subunit. This protein
binds to the superoxide byproducts of oxidative
phosphorylation and converts them to hydrogen
peroxide and diatomic oxygen. Mutations in this gene
have been associated with idiopathic cardiomyopathy
(IDC), premature aging, sporadic motor neuron
disease, and cancer. Alternate transcriptional splice
variants, encoding different isoforms, have been
characterized (23, 24).
Solute carrier family 25, member 38: LC25A38
belongs to the SLC25 family of mitochondrial carrier
proteins widely expressed in the central nervous
system (25).
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