Las 1000 instrument

The cultured cells on the dishes were washed, homogenized in lysis buffer (20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM NaN₃, 1 mM DTT, 150 mM NaCl, 0.5% Triton-X, phosphatase inhibitor cocktail (SIGMA P5726)), and centrifuged at 10,000×g for 5 min. The protein concentrations of these supernatants were measured by the Bradford method (Bio-Rad protein assay; Nippon Bio-Rad Laboratories, Tokyo, Japan). Protein samples (2 µg) were separated on 10% Tris-glycine gels (Invitrogen Japan, Tokyo, Japan) and transferred to nitrocellulose membranes (Invitrogen Japan, Tokyo, Japan). Membranes were blocked with 5% non-fat dried milk in PBS containing 0.5% Tween20 for 1 hour at 4°C, and were then incubated with primary antibodies. The membranes were incubated with peroxidase-conjugated secondary antibodies for 1 hour at room temperature. Chemiluminescence was detected by a LAS-1000 instrument (Fuji Film, Tokyo, Japan). Signals were quantified using the Fuji Image Gauge software program (ver. 3.0, Fuji Film, Tokyo, Japan).

Total proteins from supernatants, lysates and purified AfpB variants were separated by SDS-16% polyacrylamide gels and transferred to Amersham Protran 0.20 µm NC nitrocellulose transfer membrane (GE Healthcare). Protein detection was accomplished using anti-PAFB (unpublished) and anti-PAF^{27 (link)} antibodies diluted 1:1000 and 1:2000, respectively. As secondary antibody, ECL NA934 horseradish peroxidase donkey anti-rabbit (GE Healthcare) was used and chemiluminescent detection was performed with ECL™ Select Western blotting detection reagent (GE Healthcare) using a LAS-1000 instrument (Fujifilm). The experiments were repeated twice.

Briefly, 30 or 100 ng of rmCD153 (rmCD153; R&D Systems) and 100 ng of rmOPN (used as a negative control; R&D Systems) were electrophoretically separated by SDS/PAGE and blotted onto hydrated Immobilon-P PVDF transfer membranes (Merck Millipore Ltd.). Precision Plus Protein WesternC Standards (Bio-Rad) was applied to each membrane as a marker. Sera from mice immunized with the CD153#D-Alum vaccine or KLH vaccine were diluted 500-fold. The commercial anti-CD153 antibody was diluted to 0.05 μg/ml. After incubation with HRP-conjugated IgG antibodies (GE Healthcare) diluted 2000-fold for 1 h, a chemiluminescent signal visualized by Chemi-Lumi One L (Nacalai Tesque) was detected with a LAS 1000 instrument (Fuji Film) and analyzed with Multi Gauge software version 3.2.