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Cryptocephal, the Drosophila melanogaster ATF4, Is a Specific Coactivator for Ecdysone Receptor Isoform B2

Figure 2

Binding of CRC to EcR-B2 in vitro.

(A) CRC-A bound in vitro to the 17 amino acid amino-terminus of EcR-B2 and full length EcR-B2, but not to EcR-B1 or EcR-B2-E9K. This interaction was not dependent upon USP or ecdysone. Gels from two experiments are shown; the labeled protein was 35S-GAD-CRC-A in the left gel, 35S-CRC-A in the right gel. Lanes labeled “input” contained 100% of the input labeled protein. The remaining samples contained the EcR or EcR fragment shown above, and other components as indicated. GBT-B2-NS is a fusion of the DNA-binding domain of GAL4 with the amino-terminal 17 amino acids of EcR-B2. In lanes labeled B2-E9K, E9K mutant EcR-B2 or E9K mutant GBT-B2-NS was substituted 1∶1 for the corresponding wild-type protein. Full-length EcRs were precipitated with a mixture of two EcR-common region monoclonal antibodies, and GBT-B2-NS was precipitated with an antibody to the GAL4 DNA-binding domain. (B) A similar experiment, showing the binding of wild-type and mutant CRCs to full-length EcR-B2. Each incubation mixture contained the 35S-radiolabeled CRC protein listed above and the other components listed below, and precipitations were performed with the same mix of EcR common region antibodies as in (A). Wild-type CRC, CRC-R347E, and CRC-R353E bound to EcR-B2 but not EcR-B2-E9K. However, the basic-to-acidic mutation in CRC-R361E permitted binding to EcR-B2-E9K.

Figure 2