Vector map, generation, and purification of scFv-CD39 constructs. (A) Gene map of scFv-CD39 constructs in the pSectag2A vector for mammalian expression. The restriction enzymes used to insert the constructs are NotI, AscI, and XhoI. (B) Electrophoresis with 1% agarose gel. Lanes 1-3: molecular cloning of constructs using PCR amplification and double digest. (1) scFv_SCE5, AscI and NotI (821 bp); (2) scFv_MutMA2, AscI and NotI (821 bp); (3) solCD39, NotI and XhoI (1326 bp). Lane 4: DNA ladder. Lanes 5 and 6: single control digests of cloned constructs in pSectag2A. (5) targ-CD39, XhoI (7247 bp); (6) non–targ-CD39, XhoI (7247 bp). Lanes 7 and 8: triple control digests of cloned constructs in pSectag2A. (7) targ-CD39, AscI, NotI, and XhoI (821 bp for scFv_SCE5, 1326 bp for solCD39, 5100 bp for pSectag2A); (8) non–targ-CD39, AscI, NotI, and XhoI (821 bp for scFv_MutMA2, 1326 bp for solCD39, 5100 bp for pSectag2A) (C) 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis of scFv-CD39 visualized via Coomassie staining. (D) Western blot analysis of scFv-CD39s using a horseradish peroxidase–coupled anti–6xHis-tag antibody that binds to the constructs’ 6xHis-tags.