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SUPPLEMENTARY FIGURES
Figure S1. Sequence-specific binding between APC ARD and yeast two-hybrid isolates
(A) Binding of 13 independent isolates from yeast two-hybrid (Y2H) screen to WT and N507K
mutant APC ARD (Miller units indicate β-galactosidase activity in liquid assays). (B) Schematic
representation of human Trabid, with fragment corresponding to Cezanne Y2H fragment
indicated; underneath, sequence similarities between Trabid orthologs, and between human
Cezanne and Trabid. (C) Co-immunoprecipitations between WT or N507K (NK) mutant HA-
ARD from human APC and FLAG-tagged Cezanne Y2H fragment, or the corresponding
fragment from Trabid, co-expressed in 293 cells, as indicated in panels.
Figure S2. Association between Trabid and β-TrCP
Co-immunoprecipitations between HA-Trabid and FLAG-tagged WT or ΔF mutant β-TrCP
(called Fwd1 in the mouse) co-expressed in 293 cells, as indicated in panels.
Figures S3. DUB activity of Trabid
(A) Western blot from lysates of 293T cells, transfected with WT or C155A mutant HA-Trabid,
after immunoprecipitation with α-HA antibody. (B) DUB assays, with immunoprecipitates from
(A), incubated with K48- or K63-linked ubiquitin (Ub2-7); 20 µl of the Sepharose beads were
incubated with ubiquitin chains for 1 hr at 37oC.
Figure S4. Binding of Trabid to K63-linked ubiquitin
2
(A) Top, ubiquitin binding assays, with WT or mutant HA-Trabid immunoprecipitated from
transfected 293T cells, and incubated in vitro with K48- or K63-linked ubiquitin (Ub2-7);
asterisk indicates ubiquitylated protein co-immunoprecipitated with WT and C443S; background
band, IgG heavy chain. Underneath, expression levels of WT and mutant HA-Trabid. (B)
Ubiquitin binding assays, with bacterially expressed GST, or GST-tagged Trabid(1-200), and
incubated in vitro with K48- or K63-linked ubiquitin; note the strong binding preference for
K63-linked chains.
Figure S5. Subcellular distribution of Trabid
(A) Western blot of cytoplasmic and nuclear fractions of 293 cells, transfected with Trabid
siRNA 24 hrs after treatment of cells with control (L-CM) or Wnt3A-conditioned medium
(W3a-CM), probed with antibodies as indicated (the depletion was inefficient in this experiment
since only one siRNA treatment was applied). (B, C) 293 or SW480 cells, fixed and stained with
affinity-purified α-Trabid antibody (green) and DAPI (blue, to label the nuclei). (D-F) Different
human cell lines as indicated, transfected with HA-Trabid, fixed and stained with α-HA
antibody (green) and DAPI (blue). Cells were washed with PBS(+) and fixed in 1 ml pre-
warmed 4% paraformaldehyde in PBS(-) for 20 min at room temperature. Subsequently, cells
were permeabilized with 0.5% TritonX-100 in PBS(-) for 10 min, blocked with 5% normal goat
serum for 20 min, followed by incubation for 2 h with α-HA (diluted in PBS(+) containing 1%
goat serum). Cells were washed twice with PBS(+) (10 min per wash) and subsequently
incubated with Alexa488 α-rat secondary goat antibody (Molecular Probes) for 40 min and
washed 3 times with PBS(+). Coverslips were mounted on glass slides using Vectashield with
DAPI (Vector Laboratories). Fluorescence was visualized with an MRC 1024 confocal
microscope, and images were scanned at 600x magnification.
3
Figure S6. Specificity of the RNAi-mediated depletion of Trabid
(A) Semi-quantitative RT-PCR analysis, showing the levels of endogenous Trabid transcripts in
293T cells transfected with siRNAs as in Fig. 4E. (B) Western blots of lysates from 293T cells
transfected with siRNAs as in Fig. 4E, and co-transfected with HA-tagged Cezanne and Trabid,
probed with α-HA antibody. (C) Western blots of lysates from 293T cells transfected with
siRNAs as in Fig. 4E, and co-transfected with HA-tagged Trabid with silent mutations
(DsiRNA) that renders it refractory to depletion with Trabid siRNAs.
Figure S7. TCF-mediated transcription in Trabid-depleted cells stimulated with Wnt3A
TOPFLASH assays, after co-transfection of 293 cells with siRNAs and WT or mutant DsiRNA
Trabid rescue constructs as in Fig. 4F, as indicated, with or without Wnt3A stimulation as in Fig.
4B-D; underneath, Western blots, showing levels of endogenous β-catenin, and of HA-tagged
Trabid rescue constructs. For statistical significance, see Fig. 4E.
Figure S8. Epistasis analysis of Trabid function in the Wnt pathway
(A) TOPFLASH assays of 293T cells, co-transfected with siRNA against Trabid or empty
vector, or different amounts of dominant-negative FLAG-β-TrCP (ΔF). (B) TOPFLASH assays
of 293T cells as in (A), transfected with one of two different siRNAs against Trabid, or with
siRNA against Cezanne, and co-transfected with FLAG-Dvl2. (B) TOPFLASH assays in 293T
cells, transfected with Trabid siRNAs as in (A), and co-transfected with empty vector (lanes 1,
4
5), HA-Wnt3A (lanes 2, 6) or FLAG-Dvl2 (lanes 3, 7), or treated with 10 mM LiCl for 4 hrs
(lanes 4, 8).
Figure S9. Requirement of Trabid for TCF-mediated transcription in colorectal cancer cells
TOPFLASH assays of SW480 or HCT-116 colorectal cancer cells, as in Fig. 4E, after
transfection with control siRNA, one of two different siRNA against Trabid, siRNA against
Cezanne, or siRNA against β-catenin, as indicated.
Cys443
30-170129130178150651706029190176617
70-18017617919214714418213670140186129194
Putative SH3-domain binding proteinPutative mRNA splicing proteinSyntaxin 13 interacting proteinExtracellular matrix proteinDIX-domain protein, Wnt signalingUnknownBcl2-associated X proteinMelanoma antigen familyD1 proteinCCHC Zn finger, putative aspartyl proteaseGEF, BTB-POZ domain proteinPutative mitotic spindle associated proteinA20-like de-ubiquitylating enzymeCalmodulin binding protein, WD40 repeats
17311111111111
123456789101112 Cezanne13
Binding to N507K ARDhAPC
(Miller Units)Binding to WT ARDhAPC
(Miller Units)RemarksncDNA Isolates
Table 1. Results from yeast two-hybrid assay using the armadillo repeat domain (ARD) of human APC as bait and screening with a mouse cDNA library
Figure S1. Tran et al.
B
dTrabid 537 LRRALADTLHQCGHVFFTRWKEYE--MLQASMLHFTLEDSQFEEDWSTLLSLAGQPGSSLEQLHIFALAHILRRPIIVYGVKYVKSFRGEDIGYARFEGVYLPLFWDQNFCTKSPIALGYTRGHFSALVPME ::.:: :.::.:.: :.::::..: . :. :::.:.. :..:::. .::::.:::.:::: :::.:::::::::::::::: :::::: .::.::.::::::.:.:.:: ::::::::::::::::: ::hTrabid 462 LRKALHDSLHDCSHWFYTRWKDWESWYSQSFGLHFSLREEQWQEDWAFILSLASQPGASLEQTHIFVLAHILRRPIIVYGVKYYKSFRGETLGYTRFQGVYLPLLWEQSFCWKSPIALGYTRGHFSALVAME
70.5% identity in 132 aa overlap of Y2H fragment between Drosophila and human Trabid
OTU domain
1 708
Npl4-related zinc fingers (NZF domain)
200 His585
Y2H fragment
hCezanne 308 SLEEFHVFVLAHVLRRPIVVVADTMLRDSGGEAFAPIPFGGIYLPLEVPASQCHRSPLVLAYDQAHFSALVSME :::. :.:::::.:::::.: . . .. ::... : :.:::: : : .::..:.: ..::::::.::hTrabid 521 SLEQTHIFVLAHILRRPIIVYGVKYYKSFRGETLGYTRFQGVYLPLLWEQSFCWKSPIALGYTRGHFSALVAME
50.0% identity in 74 aa overlap of Y2H fragment between Cezanne and Trabid
C
A
64
51
64
28
19
28
IP a
nti
-FL
AG
Inp
uts
FL
AG
-Cez
ann
e 2
-hyb
rid
(Y
2H)
Vec
tor
WT
NK
WT
NK
WT
NK
FL
AG
-Tra
bid
OT
U (
401-
628)
HA-ARDhAPC
ARDhAPC (anti-HA)
IgG (H)
Cezanne 2H
ARDhAPC (anti-HA)
Cezanne 2HTrabid OTU
Trabid OTU(anti-FLAG)
(anti-FLAG)
Figure S2. Tran et al.
B
170.8
109.5
78.9
60.4
47.2
35.1
24.9
18.3
13.7
WT
C15
5A
Inp
ut
vect
or
K48 Ub(2-7) K63 Ub(2-7)
WB: anti-ubiquitin
Ub chains:
Ub3
Ub4Ub5Ub6Ub7
Ub2
IP anti-HA
WT
C15
5A
Inp
ut
vect
or
IP anti-HA
HA-Trabid
anti-HA
97
64
WT
C15
5A
vect
orA
64
64
51
64
64
51
FL
AG
-Fw
d1
WT
Vec
tor
HA-Trabid
Trabid (anti-HA)
Fwd1 WT
(anti-FLAG)IP a
nti
-FL
AG
Inp
uts
+ + +
FL
AG
-Fw
d1 Δ
F
Fwd1 ΔF
Fwd1 WT
(anti-FLAG)Fwd1 ΔF
Trabid (anti-HA)
Figure S3.
Figure S6.
49
95
control Trabid
vect
orW
T WT-
siRN
A
vect
orW
T
-tubulin
Trabid (anti-HA )
DNAtransfection: W
T-siR
NA
siRNA:C
120
49
TrabidCezanne
bu�e
r
cont
rol
Ceza
nne
Trab
id
-tubulin
siRNA
(anti-HA)
BA
Trabid-tubulin
RT-PCR
Cont
rol
Ceza
nne
Trab
id
- RT
siRNA
Figure S4. Tran et al.
170.8
109.578.9
60.4
47.2
35.1
24.9
18.3
13.7
5.7
170.8
109.5
78.9
60.447.2
IP anti-HA
Ub4
Ub5Ub6
Trabid
IgG(H)
(anti-Ubiquitin)
(anti-HA)
48 63 63 63Ub chains: 48 48
1%In
put
vect
or
IgG(H)
63 63 6348 48 48W
T
3xCy
sNZF
C443
S
C155
A
1 2 3 4 5 6 7 8 9 10 11 12
170.8
109.5
78.9
60.4
47.2
35.1
24.9
18.3
13.7
48 63 48 63 48 63
1%in
put
GST
GST
-Tra
bid
1-20
0
WB: anti-Ubiquitin
Ub4
Ub5Ub6Ub7
Ub chains:
A B
SW480HEK293
HA
-Tra
bid
DA
PI
97
64
64
Figure S5. Tran et al.
Trabid
-catenin
Para�bromin
con
trab
con
trab
Cytoplasmic
L-CM
siRNA:
con
trab
con
trab
L-CM W3a-CMW3a-CM
NuclearA HEK293
HEK293T
D E F
DA
PIM
erge
HEK293
rat
anti-
trab
ida�
nity
pur
i�ed
SW480
B C
Figure S7. Tran et al.
60.4
109.5
78.9
78.9
β-catenin
Trabid (anti-HA)
+ + + + + + + + + + + ++ + + + + + + + + + + +
W3a-CML-CM
α-tubulin
++ + + +FOPFLASHTOPFLASH +
++ + + ++++ + + ++ ++ + + ++
HA-Trabid transfection
WT C443S C155A WT C443S C155A
control trabidRNAi: con trab cez con trab cez
0
0.5
1
1.5
2
2.5
3
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
∗∗
∗
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Rel
ativ
e L
uci
fera
se A
ctiv
ity
Figure S8. Tran et al.
C
Figure S9.
HCT116 SW480
0
0.2
0.4
0.6
0.8
1
1 2 3 4 5 6 7 8 9 10
siRNA
con
tro
l
ceza
nn
e
Tra
bid
1
Tra
bid
2
β-ca
ten
in
con
tro
l
ceza
nn
e
β-ca
ten
in
Tra
bid
1
Tra
bid
2
64
51
A
β-TrCP-ΔF(anti-FLAG)
α-tubulin
control trabid siRNA:
400
ng
vec
200
ng
ΔF
400
ng
ΔF
200
ng
ΔF
400
ng
ΔF
0
2
4
6
8
10
12
1 2 3 4 5 6
1 2 3 4 5 6
400
ng
vec
Rel
ativ
e L
uci
fera
se A
ctiv
ity
0
1
2
3
4
5
6
7
1 2 3 4 5 6 7 8 9 10 11 12
98
50
α-tubulin
β-catenin
98hDvl2
+ + + + + + + + + +hDvl2:
siRNA: con
cez
Tra
b 1
Tra
b 2
1 2 3 4 5 6 7 8 9 10 11 12
TOPFLASHFOPFLASH
B
(anti-FLAG)
Rel
ativ
e L
uci
fera
se A
ctiv
ity
Wn
t3A
hD
vl2
LiC
l
vect
or
RNAi control RNAi Trabid
Wn
t3A
hD
vl2
LiC
l
vect
or
0
10
20
30
40
50
60
1 2 3 4 5 6 7 8
49
49
120
α-tubulin
hDvl2
Wnt3A
1 2 3 4 5 6 7 8
(anti-FLAG)
(anti-HA)
Rel
ativ
e L
uci
fera
se A
ctiv
ity
Rel
ativ
e L
uci
fera
se A
ctiv
ity