-1787
9
25 A +1 -1787 +19 hMLH1 B Acetylated H3 Enrichment -1 1 3 5 7 9 -1800 -1300 -800 -300 SW480 RKO Position from +1 B B I I + + - - SW480 B I B I + + - - RKO C hMLH1 GAPDH Ab D Methyl-H3-K4 enrichment -1 1 3 5 7 9 11 -1800 -1300 -800 -300 Position from +1 SW480 RKO SW480 B B I I + + - - RKO B I B I + + - - E GAPDH hMLH1 Ab Figure 1 F Methyl-H3-K9 enrichment Position from +1 -1 1 3 5 7 -1800 -1300 -800 -300 SW480 RKO GAPDH hMLH1 Ab SW480 B B I I + + - - B I B I + + -- G RKO
description
+1. hMLH1. -1787. +19. B. C. SW480. RKO. SW480. RKO. Acetylated H3 Enrichment. +. +. -. -. +. +. -. -. Ab. SW480. SW480. RKO. RKO. B. I. B. I. B. I. B. I. hMLH1. GAPDH. Position from +1. D. E. RKO. SW480. +. +. -. -. +. +. -. -. Ab. Methyl-H3-K4 - PowerPoint PPT Presentation
Transcript of -1787
25
A +1
-1787 +19
hMLH1
B
Ace
tyla
ted
H3
Enr
ichm
ent
-1
1
3
5
7
9
-1800 -1300 -800 -300
SW480RKO
Position from +1
BB I I
+ + - -
SW480
B IBI
+ + - -
RKO
C
hMLH1
GAPDH
Ab
D
Met
hyl-
H3-
K4
enri
chm
ent
-1
1
3
5
7
9
11
-1800 -1300 -800 -300
Position from +1
SW480RKO
SW480
BB I I
+ + - -
RKO
B IBI
+ + - -
E
GAPDH
hMLH1
Ab
Figure 1
F
Met
hyl-
H3-
K9
enri
chm
ent
Position from +1
-1
1
3
5
7
-1800 -1300 -800 -300
SW480RKO
GAPDH
hMLH1
Ab
SW480
BB I I
+ + - -
B IBI
+ + - -
GRKO
27
Figure 2
-1
0
1
2
-1800 -1300 -800 -300
Met
hyl-
H3-
K4
enri
chm
ent
B B BB I III
TSA++ ++--- -Ab-++ + + ---
hMLH1
GAPDH
C
Position from +1
D-TSA
+TSA
-1
1
3
5
7
-1800 -1300 -800 -300
Met
hyl-
H3-
K9
enri
chm
ent
Position from +1
EF
hMLH1
GAPDH
B B BB I III
TSA++ ++--- -Ab-++ + + ---
-TSA+TSA
-1
0
1
2
3
-1800 -1300 -800 -300
Ace
tyla
ted
H3
enri
chm
ent
A
GAPDH
hMLH1B B BB I III
TSA++ ++--- -Ab-++ + + ---
Position from +1
B-TSA+TSA
29
Figure 3
-1
1
3
5
7
-1800 -1300 -800 -300
Position from +1
-5-Aza-dC+5-Aza-dC
E
Met
hyl-
H3-
K9
enri
chm
ent
F
hMLH1
GAPDH
5-Aza-dC
Ab
BB II
- - - -
+ -+ -
B BI I
+ +++
+ + --
-1
1
3
5
7
-1800 -1300 -800 -300
-5-Aza-dC+5-Aza-dC
Position from +1
C
Met
hyl-
H3-
K4
enri
chm
ent D
GAPDH
5-Aza-dC
Ab
BB II
- - - -
+ -+ -B BI I
+ +++
+ + --
hMLH1
-1
1
3
5
7
-1800 -1300 -800 -300
B
BB II
- - - -
+ -+ -
hMLH1
GAPDH
5-Aza-dC
Ab
B BI I
+ +++
+ + --
Position from +1
AA
cety
late
d H
3en
rich
men
t-5-Aza-dC+5-Aza-dC
31
Fig
ure
4 Acetylated H3Enrichment
Methyl-H3-K4Enrichment
Methyl-H3-K9Enrichment
Posi
tion
from
+1
24 h
-100
0-8
00-6
00
0246 -1135 02468
-100
0-8
00-6
00
-1135 0246
-113548
h
-100
0-8
00-6
00
02468 0246
-1135
5 d
-100
0-8
00-6
00
0246
0246
-11357
12 h
drug
moc
k
Acetylated H3Enrichment
Methyl-H3-K4Enrichment
Methyl-H3-K9Enrichment
Posi
tion
from
+1
24 h
-100
0-8
00-6
00
0246 -1135 02468
-100
0-8
00-6
00
-1135 0246
-113548
h
-100
0-8
00-6
00
02468 0246
-1135
5 d
-100
0-8
00-6
00
0246
0246
-11357
12 h
drug
moc
kdr
ugm
ock
33
+ - RT
GAPDH
hMLH1
mock5-A
za-dC
+ + + + + + +- - -- -- -
mockmock
mock5-A
za-dC
5-Aza-
dC
5-Aza-
dC
dH 2O
12 h 24 h 48 h 5 d
B
mock
UMU UUUUUU U UU UMMMMM MMMMMM
5-Aza
-dC
mock
5-Aza
-dC
5-Aza
-dC
5-Aza
-dC
mock
mock
RKOSW
480
PCR dH2O
Bisulfi
te dH
2O
12 h 24 h 48 h 5 d
A
Figure 5
53
β-Actin
H3.3-HA
16 17 18 19 20 21 22 23 24WT
WT
1 2 3 4 5 8 9 10 11 12 14 15β-Actin
H3.3-HA
B
A
18 bplinker
408 bp 90 bp
H3.3 3xHA
Clone 10
Clone 16
WT
DAPIAnti-HA MergeC
D 1 3 4 116 12 13108 92 RKO 2
β-Actin
H3.3-HA
Figure 6
55
Figure 7
B
1 2 3 4 6 RKO 2
5 7 8 9 10 WT
β-Actin
H3.3-HA
13 14 15 16 17 18 19 2011 12
β-Actin
H3.3-HA
A
RKO 2
1 2 3 4 6 RKO 2
WT
8 9 10 11 12
β-Actin
H3.3-HA
C
β-Actin
H3.3-HA
RKO
WT
1 3 4 11 18 SW48
0 W
TSW
480
clon
e 20
RKO Clones
56
Figure 8
1 3 4 11 18 18 Ig
GRKO W
T
RKO Clones
SW48
0 cl
one 2
0
SW48
0 W
T
1 3 4 11 18 RKO WT
RKO Clones
Figure 8. H3.3-HA can be immunoprecipitated with anti-HA.
Whole cell (top panel) or nuclear (bottom panel) lysates were
immunoprecipitated with an anti-HA antibody. Numbers across the
top indicate clone number. Whole cell lysate from RKO clone 18 was
incubated with normal rabbit IgG as a negative control.
58
Figure 9
B
0
2468
1012
1416
18
-1000 -800 -600
Position from +1
Met
hyl-
H3-
K4
Enr
ichm
ent
SW480 cloneSW480 WTRKO cloneRKO WT
A
0
1
2
3
4
-1000 -800 -600
H3.
3 E
nric
hmen
t
Position from +1
SW480 cloneSW480 WTRKO cloneRKO WT
