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Figure S3. Generation of stably transfected C99 cell line H99C1. Human APP C99 coding sequence was cloned into pcDNA4mycHis vector (Invitrogen), which contains the Zeocin resistance gene, to generate a mammalian expression plasmid pZ-C99-mycHis. A stable cell line H99C1 was generated by transfecting pZ-C99-mycHis plasmid into HEK293 cells and selecting positive stably transfected cells with Zeocin. The stable cell line was maintained with 100 µg/ml of Zeocin. Cell lysates were analyzed by Western blot. The myc-tagged C99 proteins were detected by mouse monoclonal anti-myc antibody 9E10 and rabbit polyclonal anti APP-C-terminal antibody C20 in the H99C1 stable cells, but not in HEK293 cells. The cell lysates from pZ-C99-mycHis transiently transfected HEK293 cells (lane pZ-C99) were used as a C99myc protein size marker. β-Actin was used as control. H99C1 cells have robust expression of myc-tagged APP C99 proteins.