+
-
+
+
WB
pGEX-4T1-ERK2-Full
c
e
f
g
h
i
j
k
l
m
n
o
p
Control
Flag
V5
Actin
GST
1
2
3
4
FLAG
CRIB
SH3 BD
14-3-3 BD
EGFR BD
[ 35 S]Met-FLAG-MIG-6-1+2+3
FLAG
CRIB
SH3 BD
14-3-3 BD
[ 35 S]Met-FLAG-MIG-6-1+2
FLAG
CRIB
SH3 BD
[ 35 S]Met-FLAG-MIG-6-1
FLAG
CRIB
S35-Con.
S35-MIG-6-full
S35-MIG-6-1+2+3
S35-MIG-6-1+2
S35-MIG-6-1
GST-ERK2-pull down
S35-Con.
S35-MIG-6-full
S35-MIG-6-1+2+3
S35-MIG-6-1+2
S35-MIG-6-1
Input
V5-ERK2
Flag-MIG-6
DAPI
Flag
[ 35 S]Met-FLAG-MIG-6-full
Coomassie
Blue staining
b
C
V5
Input
B
a
Flag-MIG-6
V5-ERK2
d
Rhodamine
(V5)
+
FITC (FLAG)
-
Flag-MIG-6
V5-ERK2
IgG
Flag
IP
IgG
V5
Merge
A
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.