Colloidal gold total protein stain solution

Exosomes lysed with M-PER (10 μg) were separated through SDS-PAGE, then transferred onto polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, United States). After blocking with bullet blocking one (Nacalai Tesque, Kyoto, Japan), the membranes were incubated with rabbit polyclonal anti-AKR1B1 (1:500, sc-33219, Santa Cruz Biotechnology, Dallas, TX, United States), goat polyclonal anti-CAPG (1:100, sc-33084, Santa Cruz Biotechnology), rabbit polyclonal anti-HSP70 antibody (1:2,000, EXOAB-HSP70A-1, System Biosciences), rabbit polyclonal anti-CD63 (1:1,000, EXOAB-CD63A-1, System Biosciences), or rabbit polyclonal anti-RAB5 (ab13253, 1:1,000, Abcam, Tokyo, Japan). Immunoreactive bands were detected using enhanced chemiluminescence (EMD Millipore, Temecula, CA, United States) after incubation with horseradish peroxidase-labeled goat anti-rabbit IgG or horse anti-goat IgG (1:5,000, Vector Laboratories, Burlingame, CA, United States). Signals were detected using C-DiGit Blot Scanner (LI-COR; Kusama et al., 2018a (link)). Total proteins were stained with colloidal gold total protein stain solution according to the manufacturer’s instructions (Bio-Rad Laboratories, Hercules, CA, United States).

Recombinant bovine interferon-tau (IFNT) or interferon-alpha (IFNA) (200 ng)^{46 (link)} were mixed with Laemmil 2× buffer (4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.0% bromophenol blue, and 0.125 M Tris-HCl), and then boiled for 4 min. Samples were separated by SDS-PAGE and electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). This membrane was blocked with Block ACE (DS Pharma Biomedical, Osaka, Japan), and probed with primary antibodies against IFNT (0.3 µg/ml, Eurofins Genomics, Tokyo, Japan). Immunoreactive bands were detected using enhanced chemiluminescence (Millipore) after incubation with horseradish peroxidase-labeled goat anti-rabbit IgG antibody (0.5 µg/ml, Vector Laboratories, Burlingame, CA, USA). Total proteins were stained with Colloidal Gold Total Protein Stain solution, according to the manufacturer’s instructions (Bio-Rad Laboratories, Hercules, CA, USA).