234 J. Wu and Y.G. Zheng
Radioactive HAT assay
1.0
0.8
0.6
0.4
0.2
0
Radioactive acetyltransferase assays were conducted similarly as
previously reported (Wu et al., 2009). [14C]-labeled acetyl-CoA was
used as donor and the N-terminal 20 amino acids of histone H4(H4–
20) were used as HAT substrates. Recombinant Tip60 was expressed
in BL21 (DE3) cells.
Acknowledgments
This work was supported in part by the Georgia Cancer Coalition
Distinguished Cancer Scholar Award and National Institutes of
Health grant R01GM086717-01A2. J.W. is supported by the GSU
Molecular Basis of Disease Fellowship.
A
B
C
D
E
F
Figure 3 Activity of H4-pantetheine analogs for Tip60 inhibition.
The reaction contained 200 µm of H4-20, 10 µm of 14AcCoA and
100 nm of Tip60. The concentration of peptide-pantetheine analogs
or pantetheine is 1 mm. (A) Without inhibitor, (B) pantetheine, (C)
lysine-pantetheine, (D) H4(15–17)-pantetheine, (E) H4(14–18)-
pantetheine, (F) H4(11–22)-pantetheine.
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In the hydrolysis strategy, TFA was used to directly cleave the ad-
enosine 3′-phosphate 5′-diphosphate from the CoA motif in the
peptide-CoA conjugate (Scheme 1B). The reaction was tested for
different times (2–16 h), several temperatures (25–60°C), and vary-
ing amounts of TFA. In the final step, TFA was removed by nitrogen
blowing. After lyophilization, the mixture was dissolved in H2O and
analyzed by MALDI-MS.
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