Sul et al, Supplementary Figure S1.

Supplementary Figure S1. FAF1 is ubiquitinated by UbcH7. Each reaction contained 35S-methionine-labeled

FAF1 in addition to the indicated ubiquitination components (E1, UbcH7, UbcH8, UbcH13, parkin and Ub).

After the in vitro ubiquitination reaction, the results were obtained with autoradiography.

35S-FAF1

+ ++

FAF1

7 138

E2+ ++

E1

+ + Ub+

+

+ parkin+

96

130

72

(kDa)

Supplementary Figure S2. FAF1 mRNA expression is not decreased by parkin. Reverse transcription and

real-time PCR analysis of FAF1 mRNA in transfected SH-SY5Y cells. The primers (forward, 5′-CTGCAGGA

GTCATTAAATC-3′ and reverse, 5′-ATGGCAGGGATAAGAGAGCCC-3′). The data were calculated with

respect to the abundance of the glyceraldehyde-3-phosphate dehydrogenase mRNA.

Sul et al, Supplementary Figure S2.

Rel

ativ

e am

ou

nt

of

FA

F1

mR

NA

2.0

1.5

1.0

0.5

0.0

FAF1

FAF1

+ p

arki

n W

T

FAF1

+ p

arki

n T2

40R

Supplementary Figure S3. FAF1 mRNA expression is not increased in the ventral midbrain of Parkin-/-

mice. Reverse transcription and real-time PCR analysis of FAF1 mRNA in the ventral midbrain of Parkin-/- and

Parkin+/+ mice. The primers (forward, 5′-GGTGACTGCCATCCTGTATTTT-3′ and reverse, 5′-TGCTCTGTTG

GTGTCCTTTG-3′). The data were calculated with respect to the abundance of the glyceraldehyde-3-phosphate

dehydrogenase mRNA.

Sul et al, Supplementary Figure S3.

Rel

ativ

e am

ou

nt

of

FA

F1

mR

NA

2.0

1.5

1.0

0.5

0.0Parkin+/+ Parkin-/-

Supplementary Figure S4. Degradation of p38 by parkin WT or mutants. SH-SY5Y cells were transfected

with HA-p38 and Myc-parkin (WT and mutants), and cell lysates were analyzed by immunoblotting with an

anti-HA antibody. The transfection efficiency was confirmed using an anti-Myc antibody, and an anti-β-actin

antibody was used as a loading control.

Sul et al, Supplementary Figure S4.

HA-p38

Myc-parkin

+ + + + + +

- WT

T240

RT4

15N

HA

Myc

WB:

β-actin

G43

0DC43

1FP43

7L

+ + +

K161N

R42P

Myc-parkin

Flag-F

AF1 W

T

+

+ +- ---

Flag-F

AF1 AA

Flag-F

AF1 DD

MO

CK

Flag

Myc

β-actin

WB:

Sul et al, Supplementary Figure S5.

Supplementary Figure S5. Parkin accelerates the degradation of FAF1 independent of its

phosphorylation status. SH-SY5Y cells were transfected with Myc-parkin and Flag-FAF1 (WT, AA or DD),

and lysates were analyzed by immunoblotting with an anti-Flag antibody. Transfection efficiency was tested by

immunoblotting with an anti-Myc antibody, and the equivalency of loading was confirmed using an anti-β-actin

antibody.

CCCP (h) WB:0 3 6 12 24 36 0 3 6 12 24 36

FAF1

Tom20

Myc

β-actin

Faf1+/+ Faf1gt/gt

Myc-parkin

Sul et al, Supplementary Figure S6.

Supplementary Figure S6. FAF1 is not involved in mitophagy. Faf1+/+ and Faf1gt/gt MEFs were transfected

with Myc-parkin, subjected to carbonyl cyanide m-chlorophenylhydrazone (CCCP, 25 μM) treatment for the

indicated time periods, and total lysates were subjected to WB using specific anti-FAF1 and anti-Tom20

(Sigma) antibodies. The efficiency of transfection was confirmed using an anti-Myc antibody, and the loading

equivalency was determined by immunoblotting with an anti-β-actin antibody.

Sul et al, Supplementary Figure S7.

Supplementary Figure S7. Expression of FAF1 was increased in a time-dependent manner in response to

MPTP treatment. Lysates of the mouse ventral midbrain were prepared at indicated times after the last

injection of MPTP to analyze the FAF1 level. Left panel, Western blot of FAF1. Right panel, densitometric

analyses of band intensities normalized with respect to β-actin levels presented as mean ± SEM; **P < 0.01 and

*P < 0.05.

FAF1

β-actin

WB:21 3

3 dSaline 5 d 7 d

Saline0

2

4

6

8

3 d 5 d 7 d

MPTP (20 mg/kg)

10

**

21 3 21 3 21 3

MPTP (20 mg/kg)

Rel

ativ

e le

vel

of

FA

F1

***

Sul et al, Supplementary Figure S8.

Supplementary Figure S8. FAF1 mRNA expression is reduced in Faf1gt/gt MEFs. Reverse transcription and

real-time PCR analysis of FAF1 mRNA in immortalized Faf1+/+ or Faf1gt/gt MEFs. The primers (forward and

reverse, respectively) were targeted to exon 10 (5′-CCTCTCTTATCCCTGCCATC-3′ and 5′-ACTTCTCCGTC

ATCCACTCC-3′) or exon 12 (5′-GGTGACTGCCATCCTGTATTTT-3′ and 5′-

TGCTCTGTTGGTGTCCTTTG -3′). The data were normalized with respect to the abundance of the

glyceraldehyde-3-phosphate dehydrogenase mRNA. The results are expressed relative to the corresponding

normalized values from Faf1+/+ MEFs, and they show the means ± SEM from three independent experiments; *P

< 0.05.

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Rel

ativ

e am

ou

nt

of

FA

F1

mR

NA

Exon 10 Exon 12

Faf1+/+ Faf1gt/gt

14

12

10

8

6

4

2

0

* *

Sul et al, Supplementary Figure S9.

Supplementary Figure S9. Depletion of FAF1 protected Parkin-/- MEF cells from MPP+-induced death.

Parkin+/+ and Parkin-/- MEFs were transfected with scRNA or siRNA targeting FAF1 (siFAF1, 5′-CCACCUUCAU

CAUCUAGUC-3′) and subsequently treated with 5 mM MPP+ for 24 h. Left panel, death was determined by

assessment of LDH release. Right panel, cell lysates were analyzed by immunoblotting with the indicated

antibodies. Data are expressed as percentages of the control ± SEM; *P < 0.05.scRNA

siFAF1

scRNA

siFAF1

Parkin+/+ Parkin-/-

FAF1

parkin

β-actin

WB:

Vehicle

MPP+60

20

0

40

LD

H r

elea

se (

%)

scRNA

siFA

F1

scRNA

siFA

F1

Parkin+/+ Parkin-/-

*

MPP+ (mM)

Casp-3

FAF1

P-JNK

JNK

β-actin

WB:0 1 3 5

Sul et al, Supplementary Figure S10.

Supplementary Figure S10. JNK and caspase-3 activation is induced by MPP+ in SH-SY5Y cells. SH-

SY5Y cells were treated with different concentrations of MPP+ for 24 h, and cell lysates were analyzed by WB

using anti-FAF1, anti-JNK, anti-P-JNK, anti-Casp-3 and anti-β-actin antibodies.

Sul et al, Supplementary Figure S11.

Supplementary Figure S11. Parkin significantly reduces FAF1-induced ROS generation. SH-SY5Y cells

were MOCK transfected or transfected with Flag-FAF1 with or without co-transfection of Myc-parkin (WT or

T240R). The cells were then subjected to MPP+ (5 mM) treatment for 9 h and then incubated with or without N-

acetylcystein (NAC, Sigma), and the fluorescence intensity of DCF was measured (A.F.U., arbitrary

fluorescence units). Data show the means ± SEM from three independent experiments; **P < 0.01.

(h)0 9 9

NAC (50 μM)

**

DC

F f

luo

resc

ence

(A

.F.U

.)

MOCKFAF1FAF1 + parkin WT

FAF1 + parkin T240R

10000

5000

15000

20000

0