BA

C D

E

F

Castel_Figure S1, related to Figure 1

Rel

ativ

e m

RN

A le

vels

(2^-DD

Ct)

RT-qPCR: Hey1%

ChI

P/in

put

ChIP: Ab1-RBPJ

Sen

sitiv

ity

1-Specificity

GimmeMotifs

Transfac M00234

WT Rbpj—/—

RBPJ Phalloidin Hoechst

Hey

1

Genes associated to peaks

neg1

6

Rbm

s1

Atp

6v0a

4

Krt

9

Apo

bec3

Rps

6ka1

D

C

Castel_Figure S2, related to Figure 2

Replicate experiment ChIP:Ab1-RBPJ

On OffNotch: On Off6 hours 24 hours

Con

stan

tIn

duci

ble

p300RBPJC

onst

ant

Indu

cibl

e

On Off

24 hours

Notch: On Off

RBPJ

Con

stan

tIn

duci

ble

RBPJ

Notch: On Off24 hours

On Off6 hours

A

Castel_Figure S3

Validation with an independent RBPJ polyclonal antibody (Ab2-RBPJ)

On OffNotch: On Off

Ab1-RBPJ Ab2-RBPJ

Con

stan

tIn

duci

ble

Con

stan

tIn

duci

ble

Notch: On Off

Ab1-RBPJ

On Off

Ab2-RBPJB

CREST motif in Ab2-RBPJ only sites

RBPJ (GM) RBPJ (M00234) REST CREB ETS

FE

Castel_Figure S3F

requ

ency

D

Sen

sitiv

ity

1-Specificity 1-Specificity

Ab2-RBPJ only sites Ab1-RBPJ sites

%C

hIP

/inpu

t

ChIP: Ab1-RBPJ ChIP: Ab2-RBPJ

ChIP on REST sites

%C

hIP

/inpu

t

ChIP: Ab2-RBPJ

Hey

1

Genes associated to peaks

neg1

6

Rbm

s1

Atp

6v0a

4

Krt

9

Apo

bec3

Rps

6ka1

peaks:

A

C

RBPJ/NICD-GFP NICD-GFP only RBPJ only Constant RBPJ only Inducible

Castel_Figure S4, related to Figure 3

NICD-GFP GFP

HEYL

49kD

38kD

28kD

freq

uenc

y

freq

uenc

y

freq

uenc

y

freq

uenc

y

position (bp) position (bp) position (bp) position (bp)

B

D E

F

Castel_Figure S4, related to Figure 3

Castel_Figure S5, related to Figure 4

A

B

C