Blocking one reagent

Antibodies used for immunohistochemistry are shown in Supplementary Table 2. Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min at room temperature and then sequentially soaked in 5% sucrose/PBS for 12 h at 4 °C, 10% sucrose/PBS for 12 h at 4 °C, and 20% sucrose/PBS for 12 h at 4 °C. The cells were embedded in Tissue-Tek (Sakura Finetechnical Co., Ltd., Tokyo, Japan) and frozen. Frozen specimens were prepared at a thickness of 6 μm.
The specimens were treated with 0.2% Triton X-100 solution for 15 min at room temperature to permeabilize the cells and then treated with Blocking One Reagent (Nacalai Tesque) for 90 min to block the nonspecific adsorption of antibodies. Antibody solutions were applied to the specimens and incubated for 2 h at room temperature. After washing with PBS containing 0.05% Tween 20, the specimens were treated with fluorescently labeled secondary antibodies (1:500 dilution) for 1 h at room temperature and then washed with PBS containing 0.05% Tween 20. The cell nuclei were counterstained with 1 μg/mL Hoechst 33258 (Dojindo Laboratories, Kumamoto, Japan). The localization of secondary antibodies was analyzed with a fluorescent microscope (BX51 TRF; Olympus Optical Co., Ltd., Tokyo, Japan).

The supernatants and the cells, which were lysed with 0.3 ml of lysis buffer [150 mM NaCl, 250 mM Tris-HCl (pH 8.0), 1% NP-40, 0.1% SDS, and 1 × cOmplete (Roche Diagnostics, Mannheim, Germany)], were separated by SDS-PAGE (6 or 12%) under non-reducing or reducing conditions, followed by transfer to a PVDF membrane (Immobilon-P; Merck Millipore). The membrane was blocked with Blocking One reagent (Nacalai Tesque, Tokyo, Japan) for 30 min, followed by incubation at room temperature with the indicated antibodies. The specific bands were visualized using an enhanced chemiluminescence substrate (GE Healthcare UK Ltd., Buckinghamshire, UK) on an ImageQuant LAS4000 system (GE Healthcare). All full-length blots are shown in Figures S8 and S9.

Whole cell lysates were obtained by using lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% Triton X-100) with a protease inhibitor cocktail (Complete Mini; Roche Applied Science, Mannheim, Germany). After electrophoresis on 6–15% SDS polyacrylamide gels, proteins were transferred onto polyvinylidene difluoride membranes (Hybond-P; GE Health Care, Buckinghamshire, UK). After blocking with Blocking-One reagent (Nacalai Tesque, Kyoto, Japan) at room temperature for 30 min, membranes were incubated with the primary and secondary antibodies. The primary antibodies used were: rabbit anti-RFP (TurboFP635) polyclonal antibody (pAb) (Thermo Fisher Scientific, Fremont, CA, USA), mouse anti–alpha-smooth muscle actin (α-SMA) monoclonal antibody (mAb) (Abcam, Cambridgeshire, UK), rabbit anti-CDH1 mAb, rabbit anti-cytokeratin 20 (CK20) mAb (Cell Signaling Technology, Danvers, MA, USA), and mouse anti–β-actin mAb (Sigma-Aldrich). The secondary antibodies used were: horseradish peroxidase–conjugated antibodies against rabbit IgG or mouse IgG (GE Healthcare). Immunoreactive bands were detected using chemiluminescence substrate (ECL Plus; GE Healthcare).

The 96-well plates (Nunc MaxiSorp, Thermo Fisher Scientific) were coated with 2 μg/ml of RBD-W or RBD-O for the capture of antibodies. After blocking with BlockingOne reagent (Nacalai), the plates were incubated with serially diluted plasma or monoclonal antibodies. RBD-specific IgG antibodies were detected using horseradish peroxidase–conjugated goat anti-human IgG (Southern Biotech) with SureBlue TMB substrate (KPL). The absorbance at 450 nm was measured with a microplate reader (ARVO X3, PerkinElmer). EY6A control antibody was included on each plate for plasma samples to convert OD values into relative antibody concentrations.

Cells were plated on Matsunami Micro Cover Glass (Matsunami, Osaka, Japan), fixed with 4% paraformaldehyde solution and permeabilized with 0.1% Triton X‐100 containing Tween20. Then, they were stained with anticytochrome c antibody (12963; 1 : 300, Cell Signaling Technology, Danvers, MA, USA) and visualized using anti‐mouse IgG H&L secondary antibodies (Alexa Fluor 488; Invitrogen, Waltham, MA, USA) in Blocking One reagent (Nacalai Tesque, Kyoto, Japan). The nuclei were stained with DAPI Fluoromount‐G.