The ecdhMUC1 protein released from Ad-sig-ecdhMUC1/ecdCD40L vector-infected cells forms functional trimers and activates DCs. (A) Induction of IFN-γ secretion from bone marrow-derived DCs induced by exposure to the Ad-sig-ecdhMUC1/ecdCD40L vector. Supernatant medium collected from DCs derived in vitro from hMUC1.Tg mice after exposure to the Ad-sig-ecdhMUC1/ecdCD40L vector or to the Ad-sig-ecdhMUC1 vector and then analyzed for the levels of IFN-γ. (B) Induction of IL-12 secretion from bone marrow-derived DCs induced by exposure to the Ad-sig-ecdhMUC1/ecdCD40L or the Ad-sig-ecdhMUC1 vectors. The same procedure outlined for panel A was carried out, except that the supernatant medium was analyzed for IL-12. (C) Nondenaturing gel analysis of molecular weights of the ecdhMUC1/ecdCD40L protein. A construct was created in which a His tag was placed at the carboxyl terminal end of the CD40L, and an HSF1 trimeric stabilization domain was added between the ecdhMUC1 and ecdCD40L domains. After release from vector-infected cells, the protein was purified using a His tag column, concentrated, and added to a nondenaturing gel. The protein in the lane labeled MUC1/CD40L trimer was added to the nondenaturing gel without treatment. The protein in the lane labeled MUC1/CD40L monomer was first treated with the denaturing conditions before loading on the gel. Molecular weight markers are given in the extreme right lane.