lysate
DESCRIPTION
N.R. Sera IP. PEN-2 IP. APH-1 IP. NCT IP. PS1 IP. Lysate. (SHSY-5Y cells). TMP21. 17. TMP21. (Mouse brain). 17. Flag- TMP21. 17. (HEK293 cells). Mr. (kDa). Marker (kDa):. 29. 69. 150. 200. 443. 34. TMP21. NCT. 105. 34. PS1-NTF. 34. APH-1. 17. PEN-2. - PowerPoint PPT PresentationTRANSCRIPT
Lys
ate
PS
1 IP
NC
T IP
AP
H-1
IP
PE
N-2
IP
N.R
. Ser
a IP
TMP21
(SHSY-5Y cells)17
Flag-TMP21
(HEK293 cells)17
(Mouse brain)
TMP2117
Fraction No. 1 2 3 4 5 6 7 8 9 10 11 12
NCT
PS1-NTF
PEN-2
TMP21
APH-1
29 69 150 200 443Marker (kDa):
17
34
34
105
34
Mr.(kDa)
Native cells PEN-2 Knockdown cells PS1 & PS2 double Knockout cells
670 440 200 150 30Mr. (kDa):
TMP21
APH-1
NCT
PEN-2
PS1-FL
PS1-NTF
670 440 200 150 30670 440 200 150 30
TMP21
NCT
GM130
matureimmature
1 2 3 4 5 6 7 8 9 10 11 12
biotinylation
lysate IP unboundwith avidin lysate IP unboundwith avidin
no biotinylation
CHAPSO solubilizedMembranefraction
Conditioned media
Microsomal membranes
A4
0 p
ep
tid
e c
on
tro
l
A4
2 p
ep
tid
e c
on
tro
lAA
TMP21
AA
AA
Aβ assays inwhole cell
Aβ assays in cell free/ -secretasepreps
Em
pty
ve
cto
ro
ve
r-e
xp
res
sio
n
Tm
p2
1
ov
er-
ex
pre
ss
ion
TMP21
Co
ntr
ol
siR
NA
TM
P21
siR
NA
A
0
100
200
A
pro
du
ctio
n (
fmol
/ml)
mockp24a over-expression
A40 A42
Em
pty
vec
tor
over
exp
ress
ion
p24
a ov
erex
pre
ssio
nPS1-NTF
NCT
APH-1
p24a
TMP21
PEN-2
MW
34
105
34
16
17
17
7 Aβ assays in cell free/ -secretasepreps
Aβ assays inwhole cell
7
1 2 3 4 5 6 7
Compound E + - - - - - -PS1/PS2 + + + - + - +
TMP21-Flag Untransfected TMP21-Flag
Cell type HEK HEK HEK PS1/PS2 PS1/PS2 PS1/PS2 PS1/PS2 293 293 293 D-KO WT D-KO WT
Mr(kDa)
23
17
7
Full-lengthTMP21
Chase Time (min): 0 30 60 120 0 30 60 120 0 30 60 120
Control siRNA TMP21 siRNA Mock
APPMatImm
TMP21
BACE
APP
Nct
Cell lysate Avidin precipitation
+ + + + + + -Mock Con TMP21 Mock Con TMP21 MocksiRNA
Biotinylation
23
105
105
53
MW
Neg
ativ
e
A40A42
Tmp21T
mp
21p
oo
l
Du
ple
x A
Du
ple
x B
Du
ple
x C
Du
ple
x D
A4
2 p
epti
de
A4
0 p
epti
de