Chemi lumi one

Whole-cell extracts were prepared from W-EGFP cells using lysis buffer (50 mM Tris–HCl (pH 8.0), 50 mM NaCl, 1 mM EDTA, and 1% SDS) containing phosphatase inhibitors (PhosSTOP tablet; Roche Diagnostics Corp.). After the protein concentration was determined using a BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA), 5 μg of the whole-cell extract was subjected to SDS–polyacrylamide gel electrophoresis, followed by electrophoretic transfer to polyvinylidene fluoride membranes (Thermo Fisher Scientific). The membranes were incubated for 30 min at room temperature in Blocking One (Nacalai Tesque). The membranes were treated with primary antibodies in a reaction buffer (10% Blocking One in Tris-buffered saline; 10 mM Tris–HCl (pH 7.4), 150 mM NaCl, 0.05% Tween 20) overnight at 4 °C, and thereafter with horseradish peroxidase-conjugated secondary antibodies in the reaction buffer for 1 h at room temperature. The primary and secondary antibodies used in this study are listed in Supplementary Table 6. Washed membranes were developed using Chemi-Lumi One (Nacalai Tesque), and the images were captured using a lumino image analyzer (ImageQuant LAS 500, GE Healthcare, Buckinghamshire, UK). All images were quantified using Image Lab 6.0.0 software (Bio-Rad).

The aggregates of GM1-Glc-SFNPs and anti-GM1 antibodies in sera from patients with GBS were collected, washed three times with PBS, and then, dispersed in PBS. The dispersed solution was analyzed using SDS-PAGE and a 10% polyacrylamide gel stained with silver under reducing conditions or an 8% polyacrylamide gel under non-reducing conditions according to the typical procedure. Then, typical western blotting was performed to transfer proteins from the gel to a PVDF membrane. The membrane was then blocked with 5% skimmed milk in PBS with 0.05% Tween 20 (PBS-T) for 1 h. After washing with PBS-T three times, the membrane was incubated in a solution of HRP-conjugated anti-human IgG antibody (goat) and 5% skimmed milk in PBS-T (1:5000) for 1 h at room temperature. The gel was then developed using Chemi-Lumi One (Nakalai Tesque).

SDS–PAGE and western blotting were carried out using a Bio-Rad Mini-Protean III electrophoresis apparatus. Chemiluminescent signals generated with Chemi-Lumi One (nacalai tesque) or ECL Prime (GE Healthcare) were detected with an LAS4000 imaging system (Fuji Film). All experiment procedures were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Use Committees of Kyoto University or Keio University.

Cell and virion lysates, and immunoprecipitates were fractionated by SDS-PAGE, transferred to a PVDF membrane (Millipore), and blocked with 4% milk in PBS containing 0.1% Tween 20. Subsequently, membranes were incubated with the primary antibody, biotin-conjugated anti-mouse IgG (Sigma-Aldrich), streptavidin-conjugated horseradish peroxidase (Sigma-Aldrich), and developed using Chemi-Lumi One (Nacalai Tesque). Signals were visualized by a VersaDoc 5000 Imager (Bio-Rad Laboratories).