SUPPLEMENTARY MATERIAL
Yeast two hybrid screening
Full length human WT DJ-1 cDNA was cloned into pEG202 (EcoRI/NotI) in fusion with
LexA DNA-binding domain to be used in yeast two hybrid screening. The following
oligonucleotides were used:
DJ-1 F1 AAGAATTCGCTTCCAAAAGAGCTCTGGT
DJ-1 R1 AAGCGGCCGCCTAGTCTTTAAGAACAAGTGG
The resulting fusion construct pEG202-DJ-1 was used as a bait to screen a human foetal
brain cDNA library cloned into the galactose-inducible expression vector pJG4-5 in order
to identify DJ-1 interactors that are expressed in the nervous system.
Approximately 107 transformants were screened. Positive interaction between bait and
fish protein resulted in transcription of LacZ and Leu2 reporters, thus allowing growth in
the absence of leucine and blue staining on X-Gal plates. Strength of interaction was
evaluated by beta-Gal staining. LexA-Rrs1 was used as negative control for DJ-1
interaction.
225 clones with potential stronger interaction were isolated and further studied. Together
with a large number of clones for DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 (Ddx41;
NM_016222) and Death-associated protein 6 (Daxx; NM_001350), we isolated three
independent clones encoding for almost the entire Open Reading Frame of TRAF and
TNF Receptor Associated Protein (TTRAP; NM_016614). The interaction between
TTRAP and the DJ-1 bait was further validated in yeast taking advantage of the use of
pJG4-5-Daxx and pJG4-5-Ddx41 as positive controls since their interactions with DJ-1
have been formerly proved in Y2H and in vivo.
Constructs
For cloning TTRAP and DJ-1 open reading frames into mammalian expression vectors
the following oligonucleotides were used:
1) Cloning TTRAP into pCDNA3-2XFLAG and pCS2-6XMYC (EcoRI/XbaI):
FLAG hTTRAP fwd atatagaattcgagttggggagttgcctg
hTTRAP myc rev gcgcgctctagattacaatattatatctaagttgca
hTTRAP myc fwd atatagaattcaatggagttggggagttgcc
2) Cloning DeltaN- and DeltaC- TTRAP into pCDNA3-2XFLAG (EcoRI/XbaI):
DeltaN-TTRAP fwd ATATAGAATTCGAAGATACTCAGCAAGAAAATG
DeltaC-TTRAP rev GCGCGCTCTAGATTAAGATGGGCTGATTTTAGAAGT
3) Cloning WT, M26I and L166PDJ-1 into pCS2-6XMYC (EcoRI/XhoI):
DJ-1 F1M aagaattcagcttccaaaagagctctggt
DJ-1 R1M aactcgagctagtctttaagaacaagtgg
For TTRAP knock-down two oligonucleotides targeting different regions of TTRAP
coding sequence were selected:
si-TTRAP T1: 5’-GAAGGATATTTCACAGCTA-3’
si-TTRAP T4: 5’-GCAGGAGATACAAATCTAA-3’
The hairpin-encoding oligonucleotides were synthesized along with the loop sequence
TTCAAGAGA and the BamHI/XhoI restriction sites for cloning into the pSuperior
vector (Invitrogen).
qPCR
The following oligonucleotides were used for quantitative real time PCR:
TTRAP fwd GAACGACTGGGAGATGGAAAG
TTRAP rev CTTCATTGGTTAGGTCAACATAGG
DJ-1 fwd GAGACGGTCATCCCTGTAG
DJ-1 rev CATCTTCAAGGCTGGCATC
beta-actin fwd CGCCGCCAGCTCACCATG
beta-actin rev CACGATGGAGGGGAAGACGG
GAPDH fwd TCTCTGCTCCTCCTGTTC
GAPDH rev GCCCAATACGACCAAATCC
Cell culture media
HEK-293T (human embryonic kidney) cells were grown in DMEM (GIBCO)
supplemented with 10% foetal bovine serum (SIGMA-ALDRICH), 100 IU/ml penicillin
and 100 µm/ml streptomycin (SIGMA) at 37°C in a humidified CO2 incubator.
Human neuroblstoma SH-SY5Y cells were maintained in culture in E-MEM : F-12 with
15% fetal bovine serum, 2mM glutamine, 1% non-essential aminoacids, 100 µg/ml
penicillin and 100 µg/ml streptomycin (Invitrogen). SH-SY5Y stable cell lines were
maintained in culture under the same condition with medium supplemented with
150 µg/ml of Geneticin. Selecting drug was removed prior each experiment.
Pull down and immunoprecipitation.
For in vitro binding, HEK 293T cells were lysed in pull down lysis buffer (150 mM
NaCl, 50 mM TRIS pH 7.5, 1% NP40, 10% glycerol), supplemented with complete
EDTA-free protease inhibitor cocktail (Roche Diagnostics) for 30 minutes at 4°C.
Lysates were cleared at 15,000 g for 20 minutes. Pull down was performed for 2 h at 4°C.
For co-immunoprecipitation experiments, cells were lysed in IP buffer (Posphate
Buffered Saline (PBS), 0.5% Triton X-100), supplemented with complete EDTA-free
protease inhibitor cocktail (Roche Diagnostics) and 1mM N-ethyl-maleimide. Washes
were performed as following: two washes in IP buffer plus 500 mM NaCl, two washes in
lysis buffer and one wash in PBS.
For co-immunoprecipitation of endogenous DJ-1 in SH-SY5Y cells, a lysis buffer
containing 150 mM NaCl, 50 mM Hepes pH 7.5, 1mM EDTA, 0.1% Tween-20, 10%
glycerol was used. Washes of immunoprecipitated proteins were performed with the
same buffer.
For western blot analysis, samples were resolved on 10-12% SDS-PAGE as necessary
and proteins were transferred to nitrocellulose membrane (Schleicher & Schuell).
Membrane was blocked with 5% non-fat milk in Tris Buffer Saline Solution (TBST),
then incubated with primary antibodies overnight at 4°C or at room temperature for 2
hours. Proteins were detected by horseradish peroxidase-conjugated secondary antibodies
(DakoCytomation) and enhanced chemiluminescence reagents (GE Healthcare).
Immunocytochemistry and immunohistochemistry
For immunofluorescence experiments cells were fixed in 4% paraformaldehyde directly
added to culture medium for 10 minutes, then washed with PBS two times, treated with
0.1M glycine for 4 minutes in PBS and permeabilized with 0.1% Triton X-100 in PBS for
another 4 minutes. After washing with PBS and blocking with 0.2% BSA, 1% NGS,
0.1% Triton X-100 in PBS (blocking solution), cells were incubated with the indicated
antibodies diluted in blocking solution for 90 minutes at room temperature. After washes
in PBS, cells were incubated with labeled secondary antibodies for 60 minutes. For
nuclear staining, cells were incubated with 1g/ml DAPI for 5 minutes. Cells were
washed and mounted with Vectashield mounting medium (Vector).
For immunohistochemistry, IHC-blocking solution (10% NGS, 1% BSA, 1% fish gelatin
in PBS) was used to block unspecific background (for 1 hour at room temperature) and
for primary antibody (overnight at room temperature) and secondary antibody (2 hours at
room temperature) staining. The following antibodies were used: anti-TTRAP (1:100)
and anti-tyrosine hydroxylase (Chemicon) (1:1000). Images were collected using Leica
confocal microscope.