GFP silencing reverted cells
X
GFP silenced cells
Vectors employed
GFP expressing cells
Sf21 cell
Figure 1A
Experimental line-1
Experimental line-2
Control line
a.
b.
c.
f.
1 2 3
1 2
i
ii
d. 1 2 3
21 nt
1 M 2
14 kDa
gfp shRNA (49 bp)EcoRI19 bp 9 bp 19 bp
23 nt
PCR
RT-PCR
-actin loading control
Figure 1B
0
4000
2000
60001 2 3 4e. i
ii
iii
mRNA
Total RNAloading control
KpnI
gfp shRNA Forward primer
gfp shRNA Reverse primer
800
1600
2400
0
GFP expre
ssing lin
e
FHVB2-
C17
line
GFP silen
cing l
ine
FHVB2 e
xpre
ssin
g lin
e
Rel
ativ
e in
ten
sity
of
siR
NA
Total RNA loading control
siRNA
Figure 3
(ii)
(iii)
1 2 3 4
(i)
Figure 4
(i)
(ii)
(iii)
13 14 15 16 17 18 Catalase
Aldolase
BA C 1 2 3 M kDa
160
110
73
D 10 11 12 13 14 15 16
17 18 19 20 21 22 23
E
23mer
M 1 2
21mer
0
200 400 600
8001000
120014001600mAU
0.0 5.0 10.0 15.0 20.0 ml
14 1615
0.0 5.0 10.0 15.0 20.0 0
200
400
600
800
1000
1200
1400
1600mAU
A
25.0 ml
Bi
ii
10 11 12 13 14 15 16 17
18 19 20 21 22 23 +ve
C
1514 16
14 15 16 Pool W. Probe
- FHVB2
-DCR-1 PAZ
Figure 5
Figure 6
A
SD (Leu-Trp-His-)SD (Leu- Trp-)
(v) (vi)
YPD
(ii) (iii)
SD (Trp-) SD (Leu-)
(iv)(i)
Scheme
pGADT7+
pGBKT7
pGBKT7
pGA
DT
7 AH109AD (REP)+BD(REP)
AD
(EN
DO
)B
D(F
HV
B
2)
pGBKT7
FHVB2
AD
(PA
Z)
BD
(FH
VB
2)
B (i) (ii)
M 1 2 3 1 2 3 4 M 5 6 116 66 45 35 25
18
(iii)M 1 2 3
INPUT ELUTES FLOW THROUGH FT IEL
Figure 7
M 1 2 3 4 5 6 7
INPUTB GST-FHVB2
MBP-DCR-2-PAZ MBP
EL ELFT FT
11881
52
36
kDa
AAH109
pGAD + pGBD
pGADAD(DCR2PAZ) +BD (FHVB2)
pGBD(FHVB2)
AD(Rep)+ BD(Rep)
pGBD
AD(DCR2 PAZ)+ pGBD
(i) Scheme (ii) SD (Leu-Trp-) (iii) SD (Leu-Trp-His-)
Full Length FHVB2 (107aa)
FHVB2 – C17 (90aa)
N
N
C
C
Figure 8
A
B
C
Sf21 cell line GFP expressing GFP silenced
FHVB2- GFP reverted FHVB2- C17 transfected
00.20.40.60.81.01.2
GFP
GFP silen
ced
GFP reve
rted
FHVB2-
C17
Rel
ativ
e le
vel o
f gf
p m
RN
A
%
%%%
%
( )
( )
( ) ( )
( )
Figure 9
1 2 M 3 4 5 M 6 7
B
INPUT ELUTE F T
(i)
(i) Scheme (ii) SD (Leu-Trp-His-)
A
(iv) SD (Leu-Trp-His-)(iii) Scheme
AD+BD
BD
ADBD(FHV-B2)
AH109
AD(REP)+BD(REP) AD-
DCR1(PAZ)+BD(FHVB2-C17)
AD(PAZ) DCR1+BD(FHVB2)
AD+BD
BD
ADBD(FHV-B2)
AH109
AD(REP)+BD(REP) AD-
DCR2(PAZ)+BD(FHVB2-C17)
AD(PAZ) DCR1+BD(FHVB2)
ELUTE FTINPUT
kDa M 1 2 3
(ii)
36
52
81
118
PAZ
2249 amino acidsN-terminal C-terminal
544aa 606aa 825aa
DUF 283
920aa
HELICASE-C
510aa 1096 aa 1218 aa 1706 aa
RNase III
1919 aa
dsRNA bd
22412175aa aa
MBP-PAZ MBP-ENDONUCLEASE
dm Dicer-1
dm Dicer-2 PAZ
1715 amino acidsN-terminal C-terminal
535aa 554aa 659aa
HELICASES
1aa 838aa 945aa 1187aa
RNase III
1642aa
dsRNA bm
1710aa1646aa
MBP-PAZ
dsRNA bd
A.
B.
C.
Supplementary Figure 1
dm Dicer-1-PAZdm Dicer-2-PAZ
dm Dicer-1-PAZdm Dicer-2-PAZ
dm Dicer-1-PAZdm Dicer-2-PAZ
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