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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.

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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

Fraction No. 1 2 3 4 5 6 7 8 9 10 11 12


670 440 200 150 30

670 440 200 150 30

670 440 200 150 30

TMP21

APH-1

NCT

PS1-FL

PS1-NTF

PEN-2

Mr. (kDa):

Native cells PEN-2 Knockdown cells PS1 & PS2 double Knockout cells


no biotinylation

biotinylation

IP

IP

lysate

unbound

lysate

unbound

with avidin

with avidin

1

2

3

4

5

6

7

8

9

10

11

12

TMP21

mature

immature

NCT

GM130


Empty vector

over-expression

Tmp21

over-expression

Ab40 peptide control

Ab42 peptide control

TMP21

Ab40

Ab42

Aβ assays in

whole cell

Conditioned

media

Ab40

Ab40

Ab42

Ab42

Microsomal

membranes

Aβ assays in cell

free/ g-secretase

preps

CHAPSO

solubilized

Membrane

fraction


Control siRNA

TMP21 siRNA

TMP21

Ab


Empty vector

overexpression

p24a

overexpression

MW

p24a

17

mock

p24a over-expression

TMP21

17

200

34

PS1-NTF

Ab production (fmol/ml)

100

NCT

105

34

APH-1

0

Ab40

Ab42

16

PEN-2

7

Aβ assays in cell

free/ g-secretase

preps

7

Aβ assays in

whole cell


TMP21-Flag Untransfected TMP21-Flag

PS1/PS2 + + + - + - +

Compound E + - - - - - -

Cell type HEK HEK HEK PS1/PS2 PS1/PS2 PS1/PS2 PS1/PS2

293 293 293 D-KO WT D-KO WT

1 2 3 4 5 6 7

Mr

(kDa)

23

17

7

Full-length

TMP21


Control siRNA TMP21 siRNA Mock TMP21-Flag

Mat

APP

Imm

Chase Time (min):

0 30 60 120 0 30 60 120 0 30 60 120


Cell lysate TMP21-Flag

Avidin precipitation

siRNA

Biotinylation

Mock Con TMP21

Mock Con TMP21 Mock

+ + + + + + -

MW

Nct

105

105

APP

BACE

53

TMP21

23


Tmp21pool TMP21-Flag

Duplex A

Duplex C

Duplex B

Duplex D

Negative

Ab40peptide

Ab42peptide

Tmp21

Ab40

Ab42