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Fig. 8. Transfer of POMC from CPE to SgIII and CgA. (A) Effect of CgA, SgIII and MBP on POMC-binding capacity of CPE or SgIII. GST-CPE 23-477 or GST-SgIII 23-471 was incubated with the AtT-20 cell extract to fully bind POMC molecules. POMC-bound GST-CPE or GST-SgIII was incubated with increasing doses of recombinant CgA, SgIII or MBP, as indicated by bold figures on the top panel. GST-CPE or GST-SgIII was then pulled down with glutathione beads, and subjected to SDS-PAGE for POMC immnoblotting. In lane 9, one-twentieth of AtT-20 cell extract was run as a control. (B) In vitro pull-down of SgIII or CgA by anti-ACTH antibody. After POMC-bound GST-CPE was incubated with recombinant SgIII or CgA, POMC was pulled down by the anti-ACTH antibody. The precipitates were subjected to SDS-PAGE and immunoblotting for SgIII and CgA. Upper panel shows SgIII bound to POMC, and lower panel shows CgA bound to POMC. As a control IP experiment, preimmune serum was used instead of the anti-ACTH antibody (left panels shown as Control IP).
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