A

B

Mer

ge

a b c d

e f g h

i j k l

m n o p

Flag-MIG-6V5-ERK2

Flag-MIG-6V5-ERK2Control

DA

PI

FIT

C (

FLA

G)

Rho

dam

ine

(V5)

IgG

Flag

IgG

V5

IP

Input

V5

Flag

Flag

V5

Actin

WBFlag-MIG-6 - - + +V5-ERK2 - + - +

CRIB SH3 BD 14-3-3 BD EGFR BD

CRIB

FLAG

FLAG

CRIB SH3 BDFLAG

CRIB SH3 BD 14-3-3 BDFLAG

[ 35 S]Met-FLAG-MIG-6-full

[ 35 S]Met-FLAG-MIG-6-1+2+3

[ 35 S]Met-FLAG-MIG-6-1+2

[ 35 S]Met-FLAG-MIG-6-1

1 2 3 4

GSTpGEX-4T1-ERK2-Full

S35

-Co

n.

S35

-MIG

-6-f

ull

S35

-MIG

-6-1

+2

+3

S35

-MIG

-6-1

+2

S35

-MIG

-6-1

S35

-Co

n.

S35

-MIG

-6-f

ull

S35

-MIG

-6-1

+2

+3

S35

-MIG

-6-1

+2

S35

-MIG

-6-1

GST-ERK2-pull down

CoomassieBlue staining

Input

C

Supplemental Figure 6. MIG-6 interacts with ERK2 via its SH3 binding domain. (A) For reciprocal immunoprecipitation, 293T cells were transfected with Flag-MIG-6 and/or V5-ERK2.Cell lysates were immunoprecipitated and subsequently immunoblotted with antibodies, as indicated. (B) A schematic of the mutants of MIG-6 for either in vitro translation or immunoprecipitation and GST-fused ERK2 proteins used in the mapping studies (top). In vitro translated 35S-labeled MIG-6 deletion mutants were incubated with GST-ERK2 and the bound proteins were subjected to SDS–PAGE followed by autoradiography (bottom). (C) Ishikawa cells were seeded on coverslips and transfected with the indicated expression plasmids. The permeabilized Ishikawa cells were fixed and incubated with antibodies against FLAG and V5, which were followed by Alexa Fluor 555 donkey anti-rabbit and Alexa Fluor 488 donkey anti-mouse treatment. The fluorescence signal was analyzed using confocal microscopy.