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Supplementary Figure S1 Change of reporter activity levels after actinomycin D treatment. HEK293T cells were transiently transfected with the reporter and incubated with actinomycin D for the indicated times. - PowerPoint PPT Presentation
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• Supplementary Figure S1
(A) Change of reporter activity levels after actinomycin D treatment. HEK293T cells were transiently transfected with the reporter and incubated with actinomycin D for the indicated times.
(B) Schematic presentation of deletion constructs of mcry1 3’UTR and their reporter activity.
(C) Change of reporter mRNA levels after actinomycin D treatment. NIH3T3 cells were transiently transfected with the reporter and incubated with actinomycin D for the indicated times.
(D) Each 3′UTR in vitro transcribed with 32- α P-rUTP was subjected to in vitro binding and UV cross-linking assay with HEK 293T nuclear extract. The arrow indicates the proteins that shows differential binding.
(E) wt 3′UTR in vitro transcribed with 32- α P-rUTP was subjected to in vitro binding and UV cross-linking assay with HEK 293T cytoplasmic extract and unlabeled cold wtUTR. The arrow indicates the proteins that shows differential binding.
(F) Each 3′UTR in vitro transcribed with 32- α P-rUTP was subjected to in vitro binding and UV cross-linking assay with NIH-3T3 cytoplasmic extract. The arrow indicates the proteins that shows differential binding.
(G) wt 3′UTR in vitro transcribed with 32- α P-rUTP was subjected to in vitro binding and UV cross-linking assay with HEK 293T cytoplasmic extract and homo-oligoribonucleotide A, C, and U. The arrow indicates the proteins that shows differential binding.
(H) Alignment of conserved U-tract sequences among various species compared with between 252 and 280 in mcry1 3'UTR.
0 20 40 60 80 100 120NAT activity
pcNAT
pcNAT_wt610
pcNAT_N201
pcNAT_N401
610mcry1_3′UTR
201
401
1
CMV NAT
Woo et al_Supplementary Fig. S1
B
A
C
0
20
40
60
80
100
120
140
Act.D 0h Act.D 5h
AA
NA
T a
ctiv
ity
(%o
f A
ct.D
0h
) pcNATpcNAT_wt610
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0 2 4 6Time after Act.D [h]
pcNAT-N401pcNAT-N201
Rel
ativ
e m
RN
A l
evel
(aan
at/r
pl3
2)
homopolymer
HEK_cyto + +
A C U
+ +
mCry1_3′UTR
115
90.5
61.5
46.2
37.5
F G
Woo et al_Supplementary Fig. S1
H
N401
wt610
N201
NIH_cyto + + +
++
+
115
90.5
61.5
46.2
37.8
37.5
N401
wt610N201
HEK_nuc +
++
+
+ +
115
90.5
61.5
46.2
+
+ + +
+ +
Cold
37.5
wt610
HEK_cyto
115
90.5
61.5
46.2
D E
Sequence(5′=>3′)
Endo-mcry1 F CCT TGA AAA GCC TGG GAA AT
Endo-mcry1 R TCC GCT GCG TCT ATA TCC TC
Endo-mper2 F GCC TTC AGA CTC ATG ATG ACA GA
Endo-mper2 R TTT GTG TGC GTC AGC TTT GG
mRPL32 F AAC CCA GAG GCA TTG ACA AC
mRPL32 R CAC CTC CAG CTC CTT GAC AT
aanat F TTT GAG ATT GAG CGC GAA G
aanat R TCG AAC CAG CCC AGT GAC
mGAPDH F GCC ATC AAT GAC CCC TTC ATT
mGAPDH R GCT CCT GGA AGA TGG TGA TGG
mTBP F CAG CCT TCC ACC TTA TGC TC
mTBP R TTG CTG CTG CTG TCT TTG TT
Supplementary Table primer sequences for real time PCR