PRV US3 - figures full 111115.pptx
9
Figure 1 A B MHC I PRV US3rescue tubulin PRV gB mock PRV wt PRV infected SK cells PRV US3null PRV gD PRV US3 D C m ock PRV w t U S3null PRV US3R -20 0 20 40 60 ** * * ** *** % NK-m ediated lysis m ock PRV w t U S 3null PRV US3R 0 10 20 30 * * % NK-m ediated lysis m ock PRV w t U S3null PRV US3R 0 25 50 75 100 ** ** ** MFIR
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Transcript of PRV US3 - figures full 111115.pptx
Figure 1
mock PRV wt US3null PRV US3R-20
0
20
40
60** * *
*****
% N
K-m
edia
ted
lysi
s
A B
mock PRV wt US3nullPRV US3R0
10
20
30 * *
% N
K-m
ed
iate
d ly
sis
MHC I
PRV US3rescue
tubulin
PRV gB
mock PRV wt
PRV infected SK cells
PRV US3null
PRV gD
PRV US3
DC
mock PRV wt US3nullPRV US3R0
25
50
75
100**
****
MF
IR
Figure 2A
mock PRV wt US3nullPRV US3R0
2
4
6 *
**
** ***
MF
IR
CD300a binding
B
mock
PRV wt
PRV US3null
PRV US3Rescue
CD300a binding
MHC I
mock PRV wt US3nullPRV US3R0
5
10
15
* *
%C
D30
0a-d
epen
den
t p
rote
ctio
n f
rom
NK
cel
l-m
edia
ted
lysi
s
Mock PRV wt US3null PRV US3R0
10
20
30
40absence of KS153 (IgM)presence of KS153 (IgM)
* *
%N
K c
ell-
me
dia
ted
lys
is
Figure 3A
0 3 5 8 10
PRV US3null
PRV wt 1 µM
PRV wt 0.5 µM
PRV wt 250 nM
PRV wt 125 nM
PRV wt
mock
***
MFIR0 5 10 15
PRV US3null
PRV wt 20 µg/ml
PRV wt 10 µg/ml
PRV wt 5 µg/ml
PRV wt 2.5 µg/ml
PRV wt
mock
***
***
MFIR
CD300a binding CD300a binding
MFG-E8 Duramycin
mock PRV wt US3nullPRV US3R0
5
10
15
20
25 **
*** **
MF
IR1H6 binding
B
mock
PRV wt
PRV US3null
PRV US3Rescue
1H6 binding
CAnnexin V binding
mock
PRV wt
PRV US3null
PRV US3Rescue mock PRV wt US3nullPRV US3R
0
2
4
6
8
10 ***
**
MF
IR
Annexin V binding
D
Mock PRV wt US3null PRV US3R0
2
4
6
8
10
MF
IR
TUNEL BrdU assay TUNEL BrdU assay
mock
PRV wt
PRV US3null
PRV US3Rescue
A
mock PRV wt PRV US3null0
1
2
3
4
5*** ***
MF
IR
CD300a binding
mock PRV wt PRV US3null0
1
2
3
4 ****
MF
IR
1H6 binding
mock
PRV wt
PRV US3null
CD300a binding
1H6 binding
C
tubulin
PRV gB
mock PRV wt
PRV infected primary epithelial cells
PRV US3null
PRV gD
PRV US3
mock
PRV wt
PRV US3null
B
Figure 4
PRV US3null
tubulin
PRV gB
mock PRV wt
PRV infected SK cells
PRV D223A
PRV gD
PRV US3
Figure 5A
B
mock
PRV wt
PRV D22
3A
PRV US3n
ull0
2
4
6 ******
***
MF
IR
mock
PRV wt
PRV D22
3A
PRV US3n
ull0
4
8
12*
******
**
MF
IR
CD300a binding
1H6 binding
mock
PRV wt
PRV D223A
PRV US3null
C
CD300a binding
mock
PRV wt
PRV D223A
PRV US3null
1H6 binding
mock PRV wt PRV US3null0
2
4
6
8DMSOIPA-3
***
MF
IR
mock PRV wt PRV US3null0
2
4
6
8
10DMSOIPA3
******
MF
IR
tubulin
PRV gB
mock PRV wt
DMSO
PRV US3null
PRV gD
PRV US3
mock PRV wt PRV US3null
IPA-3
PRV infected SK cells
CD300a binding 1H6 binding
Figure 6
A
B
Figure 7
1 : 25 1 : 12 1 : 5 1 : 2,50
20
40
60
80
100 mediumisotypeIT144anti-CD16
******
******
***
*
Target:Effector-ratio
%N
K c
ell-
me
dia
ted
lys
is
(Data not shown – Rebuttal letter)
empty vector
CD300a
E59/126 IT144 KS153
Mouse monoclonal antibodies raised against human CD300a (E59/126, IT144 and KS153) recognize human CD300a and the antibody KS153 has the ability to interfere with ligand recognition of CD300a. (A) 293T cells were transfected with either a CD300a expressing plasmid or control plasmid for 48h, and subsequently the binding of monoclonal antibodies (E59/126, IT144 an KS153) was assessed by flow cytometry. X-axis of histogram plots indicates fluorescence intensity and empty profile represents appropriate isotype-matched control. (B) Jurkat cells were pretreated with either RPMI, the anti-CD300a antibody KS153 or a non-binding IgM as isotype control for 30 min, and subsequently the binding of the recombinant CD300a-Fc was assessed by flow cytometry. X-axis of histogram plots indicates fluorescence intensity and filled profile represents appropriate isotype-matched control.
A
B
CD300a-Fc
RPMI Control IgM KS153
(Data not shown – Rebuttal letter)
mock PRV wt PRV D223A-10
0
10
20
30
40
% N
K-m
edia
ted
lysi
s
The cytolytic response of PRV infected SK cells against primary porcine NK cells. SK cells were mock-infected or infected with WT PRV or kinase-inactive D223A US3 PRV (Becker strain) for 10h and subsequently incubated with IL2-primed primary porcine NK cells at a target:effector ratio of 1:25 for 4h. Viability of target cells was assessed by propidium iodide and flow cytometry, and the percentage NK cell-mediated lysis was calculated. Data represent mean + SEM of three independent experiments.
