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ER . p53. A. GST. GST-Mdm2. GST-LBD. I. I. C E2. C E2 OHT. C E2 OHT. GST. Mdm2. ER a. B. Inputs. GST-Mdm2. 1. 595. HEGO. HEGO. HE19. HE15. HE11. HE11. HE15. HE19. HEGO. A/B. C. D. E. F. HE11. A/B. D. E. F. HE19. C. D. E. F. HE15. A/B.

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ER

p53

A

GST

GST-Mdm2

GST-LBD

I

I

C E2

C E2 OHT

C E2 OHT

GST

Mdm2

ERa

B

Inputs

GST-Mdm2

1

595

HEGO

HEGO

HE19

HE15

HE11

HE11

HE15

HE19

HEGO

A/B

C

D

E

F

HE11

A/B

D

E

F

HE19

C

D

E

F

HE15

A/B

C

C

IP:Mdm2

IP:Mdm2

IP:ERa

IP: IgG

Input

Input

Input

98 -

Mdm2

Mdm2

ERa

64 -

5 6 7

1 2 3 4

D

3000

2000

Luciferase activity

*

1000

0

Gal4

Gal-GRIP

+ VP16

Gal-mdm2

+ VP16

Gal-GRIP

+ ER-VP16

Gal-mdm2

+ ER-VP16

Galmdm2

+ VP16+ p53

Gal-mdm2

+ ER-VP16+ p53

Duong et al

Figure 1


ER

Actin

Mdm2

Actin

ER

Actin

Actin

Actin

A

B

MEFWT

MEFdKO

Control

p53+Mdm2

C E2

C E2

C E2

C E2

ER

ER

C

D

CHX treatment (hrs)

0 2 4

Mdm2

ERa

ER

- + ++

w/o Estradiol

ERa

+ Mdm2

ER

Mdm2 overexpression

- +

Mdm2

- + - +

Mdm2

MG132

- - + +

Duong et al

Figure 2


Mdm2RING

ER

Mdm2 WT

Mdm2

Actin

Mdm2RING

ER

ER

Mdm2

Actin

Mdm2

ER (HE11)

Actin

ER + Mdm2RING

ER + Mdm2 WT

A

B

ER

GST-AF2

C E2

Input

GST

C

D

IP: ER

C E2 Mdm2

170 -

130 -

ER-Ub

95 -

72 -

IB: Ub

55 -

1 2 3

43 -

ER-Ub

IB: ER

4 5 6

Mdm2 - +

Duong et al

Figure 3


ER

p53

Actin

ActD

UV

ER

p53

A

C E1 E2 E3 DES

C E1 E2 E3 DES

ER

ER

p53

p53

Actin

Actin

Control

MG132

B

C

UV

ActD

Control

MG

C E2

C E2

C E2

C E2

4

3

Apoptosis (AU)

2

1

0

C

MG

D

Control

UV

C E2 OHT ICI

C E2 OHT ICI

Actin

Duong et al

Figure 4


A

B

CHX treatment (hrs)

0 2 4 6

E2+UV

ER

E2

100

p53

ERa levels (% of control)

ER

E2

E2

+UV

p53

10

6

0

2

4

Hours of chase

C

2h

4h

8h

- + - + - +

E2

ER

p53

Actin

Control

ER

p53

Actin

RITA

Duong et al

Figure 5


ER

Actin

actin

p53

pS2

A

B

C

Control

MEFdKO

Control

UV

500

E2

1200

C E2

C E2

400

1000

800

300

Relative luciferase activity

600

200

400

100

200

0

0

C

UV

C

E2

C

E2

p53+Mdm2

Control

D

900

BG1 cells

800

700

C E2

600

Relative luciferase activity

500

400

300

200

100

0

C

E2

OHT

ICI

C

E2

OHT

ICI

MCF7

BG1

Duong et al

Figure 6


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