Block ace reagent

B95a cells infected with each of the CDVs were washed once with PBS and lysed with 1% Triton X-100 in 10 mM Tris–HCl (pH 7.5), 5 mM EDTA, 1 mM dithiothreitol, 0.25 mM PMSF. Each sample was separated by 10% SDS-PAGE and blotted onto Immobilon-P membrane (Millipore, Billerica, MA, USA). After the membranes were blocked with PBS containing 4% BLOCK ACE Reagent (DS Pharma Biomedical, Osaka, Japan) and 0.05% Tween 20, they were incubated with rabbit anti-LACK antibody, rabbit anti-TSA antibody, rabbit anti-LmSTI1 antibody (described above), or rabbit anti-N protein antibody for 1 h at room temperature. After the membranes were washed, they were incubated with goat anti-rabbit IgG antibody conjugated with horseradish peroxidase (Dako Cytomation, Glostrup, Denmark) and then treated with ECL western blotting detection reagent (GE Healthcare). The reaction was visualized on an LAS 1000 Image Analyzer (Fujifilm, Tokyo, Japan).

Serum samples were separated through SDS-PAGE and were then transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). After blocking with Block Ace reagent (DS Pharma Biomedical, Osaka, Japan), membranes were incubated with goat polyclonal anti-SNX5 (1:2000, ab5983, abcam, Tokyo, Japan), rabbit polyclonal anti-SRFBP1 (1:2000, ab109598, abcam), rabbit polyclonal anti-DDB1 (1:2000, ab97522, abcam), rabbit monoclonal anti-CPSF6 (1:2000, ab175237, abcam), or mouse monoclonal anti-ACACA (1:2000, ab205883, abcam) antibody. The second western blot was conducted with rabbit polyclonal anti-SNX5 (1:2000, SAB2102260, Sigma-Aldrich, Tokyo, Japan) antibody. Immunoreactive bands were detected using enhanced chemiluminescence (EMD Millipore, Temecula, CA, USA) after incubation with horseradish peroxidase labeled anti-mouse, rabbit, or goat IgG (1:5000, Vector Laboratories, Burlingame, CA, USA). Signals were detected using C-DiGit Blot Scanner (LI-COR) and then band density was assessed with Image Studio DiGit software (version 5.2) [38 (link)]. The whole blot can be found at supplementary materials (Figures S2 and S3).