E. A. Harker et al. / Bioorg. Med. Chem. 17 (2009) 2038–2046
2045
times. During this time, a solution of 1
BSA and 10 mM DTT (15 L per well) was incubated with varying
concentrations of unlabeled peptide inhibitor in TBS-T with final
concentrations ranging from 12 nM to 200 M for 30 min. The
solutions of peptide and protein were then distributed into the
wells of the plate (30 L per well) and incubated for 1 h. Each well
was washed five times with TBS-T. The anti-MDM2 N20 antibody
Santa Cruz Biotechnology) was added to each well (100 L per
l
M hDM2 in TBS-T with 2%
a 525/25 nm filter or 633 nm HeNe laser line with a 680/30 nm fil-
ter for visualizing fluorescein and Alexa FluorÒ 647, respectively.
l
l
Acknowledgments
l
We thank Dr. Bert Vogelstein for providing HCT116 –/– cells
and Dr. Jiandong Chen for providing the hDMX expression plasmid.
This work was supported by the National Institutes of Health (GM
(
l
well) at a dilution of 1:250 in TBS-T with 1% BSA. After 1 h incuba-
tion on a microtiter shaker, the wells were washed five times with
7
4756) and the National Foundation for Cancer Research.
TBS-T, and 100 lL of anti-rabbit-IgG-HRP (Santa Cruz Biotechnol-
ogy) diluted 1:1000 in TBS-T with 1% BSA was added to each well.
References and notes
While the plate was incubated for 1 h on a microtiter shaker, the
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