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. 2010 Jun 30;285(35):26908–26915. doi: 10.1074/jbc.M110.132597

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© 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 5. — MDM2 is a bona fide E3 ubiquitin ligase for ATF3. A, purified ATF3 protein was incubated with E1, E2, MDM2, and/or ubiquitin as indicated and then subjected to immunoblotting using the ATF3 antibody. B, purified ATF3 or Δ102–139 protein was subjected to in vitro ubiquitination assay as in A. C, FLAG-tagged ATF3 or Δ102–139 protein was expressed with MDM2 in H1299 cells as indicated. Cell lysates were immunoprecipitated with the anti-FLAG antibody. D, PC3 cells were transfected with ATF3, MDM2, and/or FLAG-Ub as indicated. Cell lysates were immunoprecipitated with the ATF3 antibody and subjected to immunoblotting (IB) for ubiquitinated proteins using the FLAG antibody. E, H1299 cells were transfected with FLAG-ATF3, MDM2, and/or HA-Ub as indicated, followed by IP assays using the FLAG antibody. Ubiquitinated proteins were detected with an anti-HA antibody.