Protein Analysis of CSF and Serum

CSF and serum samples were dissolved in Laemmli buffer without 2-mercaptoethanol, boiled for 3 min, and loaded on SDS-polyacrylamide gels (194–1502, FUJIFILM Wako) [27 (link)]. After SDS-PAGE, protein bands were visualized with a Silver Stain II kit (FUJIFILM Wako). For immunoblotting, proteins separated on gels were transferred to nitrocellulose membranes. The membranes were blocked in 3% skim milk, and incubated sequentially with the following combinations of primary and secondary antibodies: anti-Tf antibody (Bethyl Laboratories, Montgomery, TX, USA) and horseradish peroxidase (HRP)-labeled anti-goat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA); anti-PDGS antibody (PA1-46023, Thermo Fisher Scientific, Waltham, MA, USA) and HRP-labeled anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA); anti-TTR antibody (ab9015, Abcam, Cambridge, CB2 0AX, UK) and HRP-labeled anti-sheep IgG antibody (#31480, BiotechnologyThermo Fisher Scientific). The protein bands were visualized with a SuperSignal West Dura Chemiluminescence Substrate Kit (Pierce Biotechnology, Rockford, IL, USA). Purified standards of GlcNAc and Man-Tf mixture were purified from CSF as described previously [15 (link)]. His-tagged TTR (89-7754-27) and L-PGDS (E-PKSH030653.10) were purchased from Elabscience Biotechnology Inc., (Houston, TX, USA) and Biomol (Hamburg, Germany)