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. 2018 Mar 21;27(3):438–455. doi: 10.1177/0963689718759473

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© The Author(s) 2018

This article is distributed under the terms of the Creative Commons Attribution 4.0 License (http://www.creativecommons.org/licenses/by/4.0/) which permits any use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage).

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Fig. 4. — Protein size-exclusion chromatography by preparative-grade Superdex 200 column. The 0.8 M NaCl-eluted protein fraction of cord blood serum was further subjected to size-exclusion chromatography by analytic Superdex 200 column. Approximately 100 mg of protein from the 0.8 M NaCl fraction was applied to the column, and 48 fractions were eluted with phosphate-buffered saline (PBS). (A) Chinese hamster ovary cells stably expressing wild-type human APP (CHO/APPwt) cells were cultured in 24-well plates and treated with 40 μL of each protein fraction for 2 h. The conditioned media were collected and analyzed by soluble amyloid precursor protein α (sAPPα) Western blot (upper panel) and ELISA (lower panel). In parallel, the 0.8 M NaCl-eluted fraction (#31) and PBS (#32) were included under the same cell culture conditions as positive and negative controls, respectively. Cell lysates were also prepared from each fraction-treated cell culture as an additional reference to evaluate sAPPα levels. (B) Protein concentration of each size fraction. (C) CHO/APPwt cells were treated with #8 to 18 size fractions prepared from 3 independent experiments, as well as the original 0.8 M NaCl fraction (#31) and PBS (#32), and then conditioned media were collected and analyzed by sAPPα ELISA. The results were presented as mean (±SD) sAPPα produced (ng/mg protein). In addition, each size fraction was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis to assess total protein fractionation (C, right panel).