1

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