H 2 O 2
BP
APEX2
+
+
+
+
+
+
+ +
+
+
-
-
-
-
-
+
+
+
+
+
+
+ +
+
+
-
-
-
-
-
+
+
+
+
+
+
+ +
+
+
-
-
-
-
-
+
+
+
+
+
+
9 µl beads
3/
9/ 1/
15
25
streptavidin-HRP
ponceau
anti-V5
-NES)
s
n
o
i
t
u
l
e
)
t
u
p
n
i
(
s
t
c
a
r
t
x
e
l
l
e
c
V5-APEX2-NES
APEX2
35
40
55
70
100
15
25
35
40
55
70
100
10
130
180
15
25
35
40
55
70
100
10
130
flowthrough
APEX2
180
15
25
35
40
55
70
100
streptavidin-HRP
streptavidin-HRP
V5-APEX2-NES
H 2 O 2
BP
+
+
+
+
+
+
+
+
+
-
-
-
anti-GFP
(CNX43-APEX2)
CNX43-GFP-APEX2
cell extracts
15
25
35
40
55
70
100
CNX43-GFPAPEX2
truncated GFP
H 2 O 2
BP
+
+
+
APEX2(GFP)
merge
DAPI
Biotin
APEX2-NES
CNX43-APEX2
control cells
3000
0
1000
2000
4000
5000
size (nt)
A
B
C
D
E
F
G
Fig. 3 Proximity-CLIP checkpoints and controls. (a–d) Control cells (growing on 6 cm plates) expressing an
APEX2-fusion protein (V5-APEX2-NES) and supplemented with biotin-phenol (BP) and hydrogen peroxide
(H 2 O 2 ) or devoid of at least one of the three components were extracted and underwent proximity biotinylation.
(a) Horseradish peroxidase (HRP)-coupled streptavidin Western blot analysis of protein biotinylation in cells
expressing an APEX2-fusion protein and supplemented with biotin-phenol (BP) and hydrogen peroxide (H 2 O 2 ),
or devoid of at least one of the three components. Ponceau stain is used as a loading control. (b) Western blot
validation of APEX2-fusion protein expression. (c and d) Validation of pull-down efficiency combined with
calibration of the optimal ratio between amounts of beads and cell extract. (c) HRP-coupled streptavidin
292
Daniel Benhalevy and Markus Hafner