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. 2008 Aug 11;182(3):531–542. doi: 10.1083/jcb.200711151

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© 2008 Ju et al.

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Figure 3. — Cosmc has ATP-binding activity. (A) Hi-5 insect cells were infected with Baculovirus encoding either Cosmc-HA or HPC4-sT-synthase plus wild-type Cosmc. Cells for Cosmc-HA and media from cell coexpressing HPC4–sT-synthase and wild-type Cosmc were harvested. The cell extracts and media were loaded on ATP-Sepharose column for chromatography and eluted with 50 mM ATP, respectively. The washes and eluates were analyzed by Western blot with anti-HA for Cosmc and anti-HPC4 for T-synthase, as indicated. (B) HPC4-sCosmc and HPC4–sT-synthase (coexpressed with wtCosmc) were expressed in Hi-5 cells and purified from the media. 3 μg of Cosmc and T-synthase were photolabeled with 8-Azido α-[³²P]ATP, analyzed on SDS-PAGE, and transferred to nitrocellulose membrane for radioautogram and Western blot with anti-HPC4. Black line indicates that intervening lanes have been spliced out.