Journal of the American Chemical Society
Page 6 of 8
transfected in Opti-MEM (Gibco) with 12 µg of plasmid and Dorothy Dorsett Brown Foundation and Helen
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
DNA using Lipofectamine 2000 (Life Technologies)
according to manufacturer’s protocol. 6 hours post-
transfection, media were changed to 10 mL DMEM
without phenol red. 72 hours post-transfection, cell
culture media were collected and centrifuged at 1,000 rpm
for 5 minutes to remove residual cells. The supernatant
was then concentrated to 100 µL using a 10 kDa MWCO
filter (Millipore). Meanwhile, cells were harvested and
lysed in 50 mM Tris pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5%
NP-40, 0.5% sodium deoxycholate. Expression of
corresponding proteins was tested by western blotting of
McLoraine Chair in Molecular Neurobiology (K.-F.L.), the
NCI Cancer Center Support Grant P30 (CA014195 MASS
core, A.S.), the Leona M. and Harry B. Helmsley
Charitable Trust grant (#2012-PG-MED002, A.S.), and Dr.
Frederick Paulsen Chair/Ferring Pharmaceuticals (A.S.).
REFERENCES
(1) Kim, D. H.; Yeo, S. H.; Park, J. M.; Choi, J. Y.; Lee, T.
H.; Park, S. Y.; Ock, M. S.; Eo, J.; Kim, H. S.; Cha, H. J.
Gene 2014, 545, 185.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
0 µg of cell lysate or conditioned medium using anti-myc
(2) Chouraki, V.; Seshadri, S. Adv. Genet. 2014, 87, 245.
(3) O'Brien, R. J.; Wong, P. C. Annu. Rev. Neurosci. 2011,
34, 185.
(4) Haass, C.; Kaether, C.; Thinakaran, G.; Sisodia, S. Cold
Spring Harb. Perspect. Med. 2012, 2, a006270.
(5) Newman, M.; Musgrave, I. F.; Lardelli, M. Biochim.
Biophys. Acta 2007, 1772, 285.
antibody. Recombinant ApoE4 (5 µg) was incubated with
either cell lysate or conditioned medium for 4 hours at
37°C in 50 mM Tris pH 8.0 with total volume of 15 µL.
After reactions were completed, 5 µL of 4×SDS loading
dye were added. Samples were boiled at 95°C for 10 min
TM
and subjected to SDS-PAGE using Bolt 4-12% Bis-Tris
Plus polyacrylamide gels (Life Technologies). ApoE full-
length protein and its proteolytic fragments were
visualized by western blotting.
(6) Cras, P.; Kawai, M.; Lowery, D.; Gonzalezdewhitt, P.;
Greenberg, B.; Perry, G. Proc. Natl. Acad. Sci. U.S.A. 1991,
88, 7552.
(7) Hardy, J.; Selkoe, D. J. Science 2002, 297, 353.
(8) Thinakaran, G.; Koo, E. H. J. Biol. Chem. 2008, 283,
29615.
(9) Mucke, L. Nature 2009, 461, 895.
(10) Yamin, G.; Ono, K.; Inayathullah, M.; Teplow, D. B.
Curr. Pharm. Des. 2008, 14, 3231.
Tau degradation in the presence of ApoE. 600 nM of
Tau were incubated with 30 nM of purified human ΔN-
HtrA1 (aa 156-480) and 600 nM of ApoE3 or ApoE4 in 50
mM Tris pH 8.0 at 37 °C. 10 µL of reaction was taken every
30 minutes, mixed with 5 µL of 4×SDS loading dye.
Samples were boiled at 95°C for 10 min and subjected to
(11) Teich, A. F.; Arancio, O. Biochem. J. 2012, 446, 165.
(12) Cummings, J. L.; Morstorf, T.; Zhong, K. Alzheimers.
Res. Ther. 2014, 6, 37.
(13) Tanzi, R. E. J. Alzheimers Dis. 2013, 33, S5.
(14) Mahley, R. W.; Weisgraber, K. H.; Huang, Y. Proc.
Natl. Acad. Sci. U.S.A. 2006, 103, 5644.
(15) Corder, E. H.; Saunders, A. M.; Strittmatter, W. J.;
Schmechel, D. E.; Gaskell, P. C.; Small, G. W.; Roses, A.
D.; Haines, J. L.; Pericak-Vance, M. A. Science 1993, 261,
921.
TM
SDS-PAGE
using
Bolt
4-12%
Bis-Tris
Plus
polyacrylamide gels (Life Technologies). Full-length Tau
protein was visualized by silver staining.
ASSOCIATED CONTENT
Supporting Information
Supplementary methods and figures are described. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(16) Strittmatter, W. J.; Roses, A. D. Proc. Natl. Acad. Sci.
U.S.A. 1995, 92, 4725.
(17) Kim, J.; Basak, J. M.; Holtzman, D. M. Neuron 2009,
63, 287.
AUTHOR INFORMATION
Corresponding Author
(18) Pitas, R. E.; Boyles, J. K.; Lee, S. H.; Foss, D.; Mahley,
R. W. Biochim. Biophys. Acta 1987, 917, 148.
(19) DeMattos, R. B.; Brendza, R. P.; Heuser, J. E.;
Kierson, M.; Cirrito, J. R.; Fryer, J.; Sullivan, P. M.; Fagan,
A. M.; Han, X.; Holtzman, D. M. Neurochem. Int. 2001, 39,
415.
*
asaghatelian@salk.edu (A.S.)
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
(20) Ikonen, E. Nat. Rev. Mol. Cell Biol. 2008, 9, 125.
(21) Puglielli, L.; Tanzi, R. E.; Kovacs, D. M. Nat. Neurosci.
2003, 6, 345.
(22) Liu, C. C.; Kanekiyo, T.; Xu, H.; Bu, G. Nat. Rev.
Neurol. 2013, 9, 106.
We thank Dr. Zhijiang Chen, Dr. Laura Tan and Professor
Paul Sawchenko for fruitful discussions. U-87 MG cell line
is a generous gift from Dr. Natalie Luhtala and Professor
Tony Hunter at the Salk Institute. We also thank
Matthew Kolar for running ABPP gels. Q.C. is a
postdoctoral fellow funded by the George E. Hewitt
Foundation for medical research. This study was
supported by the National Institutes of Health AG042985,
AG0476669, the Clayton Foundation, the Schlink
Foundation, the Gemcon Family Foundation, the Joe W.
(23) Mahley, R. W.; Rall, S. C. Annu. Rev. Genomics Hum.
Genet. 2000, 1, 507.
(
(
24) Roses, A. D. Annu. Rev. Med. 1996, 47, 387.
25) Clement-Collin, V.; Barbier, A.; Dergunov, A. D.;
Visvikis, A.; Siest, G.; Desmadril, M.; Takahashi, M.;
Aggerbeck, L. P. Biophys. Chem. 2006, 119, 170.
ACS Paragon Plus Environment