Anti mouse apn antibody

Whole serum, fractionated serum samples, or isolated plasma exosomes were incubated at 100°C for 5 minutes in Laemmli SDS sample buffer with 2-mercaptoethanol. The samples were separated with 4%-20% gradient SDS-PAGE gels (Bio-Rad, #5671095) and transferred onto nitrocellulose membranes. The membranes were blocked with Blocking One (Nacalai, #03953-95) for 60 minutes at room temperature, incubated with primary antibodies using Can Get Signal Solution 1 (TOYOBO, #NKB-201) overnight at 4°C, and incubated with secondary HRP-conjugated antibodies using Can Get Signal Solution 2 (TOYOBO, #NKB-301) for 60 minutes at room temperature. The following antibodies were used: anti-mouse APN antibody (R&D, #AF1119); anti-alphatubulin antibody (Cell Signalling, #2125S); anti-ALIX antibody (Santa Cruz, #sc-53538); anti-Tsg101 antibody (Abcam, #ab125011); anti-syntenin antibody (Abcam, #ab19903); HRP-conjugated anti-Goat IgG antibody (Thermo, #811620); and HRP-conjugated anti-rabbit IgG antibody (GE Healthcare, #NA934V).