sh-lu

c

shS

CP

1-1

*

IB: c-Myc

IB: Tubulin

shS

CP

1-2

IB: p-Ser621.0 1.8 2.5

60KD70KD

60KD70KD

1 2 3

(abnova)

sh-lu

c

shS

CP

1-1

*

IB: c-Myc

IB: Tubulin

shS

CP

1-2

IB: p-Ser62

1.0 3.1 3.360KD70KD

60KD70KD

1 2 3

IB: SCP1

(abcam)

B

C

60KD70KD

60KD70KD

1 2 3 4 5 6 7 8 9 10 11 12

NC shSCP1-1 shSCP1-2

*

IB: c-Myc

IB: Actin

IB: p-Ser62

IB: SCP1

0 20 40 60 0 20 40 60 0 20 40 60 CHX(min)

(abcam)

0 20 40 60 0 20 40 60 0 20 40 60 CHX(min)

NC shSCP1-1 shSCP1-2

*

IB: c-Myc

IB: Tubulin

IB: p-Ser62

1 2 3 4 5 6 7 8 9 10 11 12

(abnova)

Figure S1

AT

58A

S62

A

T88

A/S

62A

IB: p-Ser62 (abcam)

IB: p-Ser62 (abnova)

IB: c-Myc

WT

IB: HA-GFP

1 2 3 4

HA-c-Myc

Figure S1. Specificity of two c-Myc Ser62 phosphorylation antibodies from Abcam and

Abnova. (A). Both phosphorylation antibodies recognize the phosphorylated Ser62. HEK293T cells

were transfected and c-Myc were analyzed by Western Blotting. (B). HepG2 cells infected with

retrovirus were lysed and the protein levels were analyzed using Western Blotting. * indicatded a

non-specific protein band.. ( C) Knockdown of SCP1 prolonged c-Myc half-life. HepG2 cells were

infected with retrovirus of sh-Luc and shSCP1. The half-life of endogenous c-Myc in control or SCP1

knockdown cells was detected using a CHX chase assay. * indicated a non-specific protein band.

D

IB: c-Myc

IB: SCP1

IB: SKP2

IB: Actin

siNC siSKP2SCP1 - + - +

c-M

yc p

rote

in e

xpre

ssio

n

siNC siFBW7 siNC siSKP2

1 2 3 4 1 2 3 4

IB: c-Myc

IB: SCP1

IB: FBW7

IB: Actin

SCP1 - + - +siNC siFBW7

Figure S2

Figure S2. SCP1 affects c-Myc stability dependent on FBW7. (A). SCP1 has no significant

effect on PP2A protein level. HEK293T cells were transfected as indicated. PP2A were analyzed

by Western Blotting using PP2A specific antibody (CST #4953) and (B) the different subunits of

PP2A were analyzed by QPCR. (C). SCP1 increases c-Myc ubiquitination. HEK293T cells were

transfected as indicated. Cells were treated with MG132 for 6 hours, and ubiquitination was

analyzed using Ni-NTA-based pull-down assays. (D). Knockdown of FBW7, but not SKP2,

blocked the effect of SCP1 on c-Myc stability. HEK293T were transfected with 40nM of control

siRNA (siNC), siFBW7 or siSKP2 respectively. After 24h, FLAG-vector or FLAG-SCP1 were

transfected into cells in the indicated combinations and analyzed by Western Blotting.

C HA-c-Myc + + + + - -His-Ub - + + + + +FLAG-SCP1 - - WT DN WT DN

Ni-NTAprecipitated

WCE

IB: FLAG

IB: c-Myc

1 2 3 4 5 6

Myc(Ub)nIB: c-Myc

IB: PP2A B unit

vect

or

IB: Tubulin

IB: SCP1

SCP1-W

TSCP1-

DN

A

B

Figure S3

Rel

ativ

e m

RN

A e

xpre

ssio

n

RhoA reporter

Rel

ativ

e lu

cife

rase

act

ivity

ctrl c-MycWT

c-MycS62A

c-MycS62D

**

**

**

IB:c-Myc

RhoA

**

****

Figure S3. The transcriptional activity of c-Myc wild-type and its mutant. (A). SCP1 has no

significant effect on mRNA level of WDFY2 and PIP5K1A. HEK293T cells were transfected as

indicated. The expression of WDFY2 and PIP5K1A mRNA were analyzed by Q-PCR. (B). c-Myc

WT, S62A and S62D mediated RhoA reporter gene activity. HEK293T cells were transfected as

indicated, the promoter activity were analyzed by Dual-Luciferase assay, c-Myc protein

expression were detected by Western blot. (C,D). HEK293T were transfected as indicated, the

RNA expression of RhoA or Bax were detected by Q-PCR. The data are means ± s.d. (**

P<0.01, t-test; n=3)

A

CR

elat

ive

mR

NA

exp

ress

ion

Bax

**

**

*

D

B

Figure S4

OD

490

(nm

)O

D 4

90 (

nm)

HeLa

HT29

IB:c-Myc

IB: SCP1

IB: Actin

sh-SCP1 - + - +sh-c-Myc - - + +

IB:c-Myc

IB: SCP1

IB: Actin

sh-SCP1 - + - +sh-c-Myc - - + +

A

B

1 2 3 4

1 2 3 4

Figure S4. SCP1 affects cells proliferation in a c-Myc dependent manner. HeLa

(A) or HT29 (B) were infected with retrovirus as indicated. The cell proliferation

was detected using MTT assay, and knockdown of c-Myc and SCP1 were

analyzed by Western Blotting