Journal of the American Chemical Society
Article
loading buffer. The samples were analyzed by Western blotting as
described above. For photo-cross-linking with FRB-HA, (a) anti-human
FKBP12 antibody and anti-rabbit IgG antibody-HRP conjugate, (b)
anti-HA tag antibody (abcam) and anti-rabbit IgG antibody-HRP
conjugate, and (c) SAv-HRP were used. For photo-cross-linking with
CnA, (a) anti-carcineurin A antibody (abcam) and anti-rabbit IgG
antibody-HRP conjugate and (b) SAv-HRP were used.
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Intracellular Photo-Cross-Linking Experiments. HeLa cells
were transiently transfected with pC4-RHE plasmid (ARIAD Pharma-
ceuticals), which encodes FRB-HA, using Lipofectamine LTX
(Invitrogen). After 5 h of transfection, endogenous FKBP12 labeling
was performed by incubating the cells in serum-free DMEM
containing 11 (5 μM) at 37 °C for 18 h. Subsequently, the medium
was replaced with serum-free DMEM containing or noncontaining
rapamycin (5 μM). As a negative control, the cells were incubated with
FK506 (30 μM) and rapamycin (5 μM). The cells were then exposed
to 365 nm light by using a UV lamp (8 W) at 4 cm distance from the
top of the cell culture dish (without the lid) on ice for 20 min. The
cells were washed twice with PBS and lysed using NP-40 lysis buffer.
After centrifugation, the lysate was dialyzed against PBS with a
Spectra/Por dialysis membrane (MWCO: 3,500). Biotinylated
proteins were isolated by incubating the lysates with immobilized
NeutrAvidin protein beads (Pierce) at 4 °C for 1 h. The beads were
washed 10 times with PBS containing 0.05% Tween and boiled in
SDS-PAGE loading buffer containing 5 mM biotin. The samples were
analyzed by Western blotting using (a) anti-human FKBP12 antibody
and anti-rabbit IgG antibody-HRP conjugate, (b) anti-HA tag antibody
(abcam) and anti-rabbit IgG antibody-HRP conjugate, and (c) SAv-HRP.
(9) (a) Tsukiji, S.; Miyagawa, M.; Takaoka, Y.; Tamura, T; Hamachi,
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(10) (a) Wold, F. Methods Enzymol. 1977, 46, 3−14. (b) Chen, G.;
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(11) Another type of traceless affinity labeling based on an acyl
transfer reaction has been recently reported: Hughes, C. C.; Yang,
Y.-L.; Liu, W.-T.; Dorrestein, P. C.; La Clair, J. J.; Fenical, W. J. Am.
Chem. Soc. 2009, 131, 12094−12096.
ASSOCIATED CONTENT
* Supporting Information
Figures S1−S18, Tables S1−S3, the experimental details of the
synthesis, and complete ref 3j. This material is available free of
■
S
AUTHOR INFORMATION
Corresponding Author
■
(12) Clackson, T.; Yang, W.; Rozamus, L. W.; Hatada, M; Amara, J.
F.; Rollins, C. T.; Stevenson, L. F.; Magari, S. R.; Wood, S. A.;
Courage, N. L.; Lu, X.; Cerasoli, F.; Gilman, M.; Holt, D. A. Proc. Natl
Acad. Sci. U.S.A. 1998, 95, 10437−10442.
(13) (a) Takaoka, Y.; Tsutsumi, H.; Kasagi, N.; Nakata, E.; Hamachi,
I. J. Am. Chem. Soc. 2006, 128, 3273−3280. (b) Harvey, J. H.; Trauner,
D. ChemBioChem 2008, 9, 191−193.
ACKNOWLEDGMENTS
■
We thank ARIAD Pharmaceuticals for providing the ARGENT
Regulated Heterodimerization Kit. T.T. acknowledges the Japan
Society for the Promotion of Science (JSPS) Research Fellowships
for Young Scientists. S.T. thanks the Takeda Science Foundation
for financial support.
(14) In separate experiments, we confirmed that the hydrolytic
stability of reagents 5 and 7 in aqueous buffer solution was almost
similar (Figure S1 in SI). Thus, the different labeling efficiency was not
caused by a change in the stability of the two reagents.
(15) Although the spacer structure is identical in reagents 5 and 8,
the labeling kinetics of the two reagents was dissimilar (Figure 4c).
This suggests that the probe moiety also affects the labeling properties
to some extent.
(16) Holt, D. A.; Luengo, J. I.; Yamashita, D. S.; Oh, H.-J.; Konialian,
A. L.; Yen, H.-K.; Rozamus, L. W.; Brandt, M.; Bossard, M. J.; Levy, M.
A.; Eggleston, D. S.; Liang, J.; Schultz, L. W.; Stout, T. J.; Clardy, J.
J. Am. Chem. Soc. 1993, 115, 9925−9938.
(17) As another rare example of tyrosine modification by affinity
labeling using a bromoacetyl reactive group: (a) Haimovich, J.; Eisen,
H. N.; Hurwitz, E.; Givol, D. Biochemistry 1972, 11, 2389−2398.
(b) Pollack, S. J.; Nakayama, G. R.; Schultz, P. G. Science 1988, 242,
1038−1040.
(18) Recently, Cravatt and coworkers reported that the phenyl-
sulfonate ester group has reactivity to histidine, tyrosine, and glutamate
(and aspartate and cysteine) residues in a proteome-based labeling
method: Weerapana, E.; Simon, G. M.; Cravatt, B. F. Nat. Chem. Biol.
2008, 4, 405−407.
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