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Lysate

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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Lysate

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  1. 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)

  2. 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

  3. 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

  4. 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

  5. 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

  6. Control siRNA TMP21 siRNA TMP21 Ab

  7. 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

  8. 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

  9. Control siRNA TMP21 siRNA Mock Mat APP Imm Chase Time (min): 0 30 60 120 0 30 60 120 0 30 60 120

  10. Cell lysate Avidin precipitation siRNA Biotinylation Mock Con TMP21 Mock Con TMP21 Mock + + + + + + - MW Nct 105 105 APP BACE 53 TMP21 23

  11. Tmp21pool Duplex A Duplex C Duplex B Duplex D Negative Ab40peptide Ab42peptide Tmp21 Ab40 Ab42

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