Construction of tumor Id and CTLA-4 fusion protein.

(A) Schematic diagram of Id-CTLA4. Solid areas represent variable regions from the 38C13 tumor. Open areas represent human γ1 and κ constant regions. Checkered regions represent the human CTLA-4 sequence. (B) SDS-PAGE of Id (lanes 1 and 4), Id-GM (lanes 2 and 5), and Id-CTLA4 (lanes 3 and 6) under nonreducing (lanes 1-3) or reducing (lanes 4-6) conditions. The molecular weight is determined by marker proteins. (C) Immunoblot analysis of Id fusion proteins. Id (lanes 1 and 4), Id-GM (lanes 2 and 5), and Id-CTLA4 (lanes 3 and 6) under nonreducing (lanes 1-3) or reducing (lanes 4-6) conditions were subjected to SDS-PAGE followed by electroblotting to nitrocellulose membrane. The strips were reacted with S5A8, a monoclonal anti-38C13 Id (lanes 1-3) or mouse antihuman CTLA-4 (lanes 4-6) Abs and detected with HRP-conjugated second-step reagents.