Sc 33219

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).

Cells cultured on glass chamber slides were fixed with ice-cold acetone and blocked in phosphate buffered saline with 10% bovine serum albumin and 10% goat serum for 1 h at room temperature. Immunofluorescent detection was carried out for 1 h at room temperature with commercially available antibodies: mouse (4 μg/ml, sc-514024, SantaCruz Biotechnology, Santa Cruz, CA) and rabbit (10 μg/ml, nbp188641, Novus Biologicals, Abingdon, UK) anti-SLC6A12; rabbit (2 μg/ml, sc-33219, SantaCruz Biotechnology) and goat (1 μg/ml, sc-17732, SantaCruz Biotechnology) anti-AKR1B1; mouse anti-developmental myosin heavy chain (dMyHC) (40 μg/ml, RMMy2/9D2, Leica Biosystems, Nussloch, Germany). The corresponding Alexa594—and Alexa488-labeled secondary antibodies (ThermoFisher Scientific) were added. After mounting in Fluoromount G (Southern Biotech, Alabama, USA), digital photography was performed on a Zeiss Axiophot microscope (Zeiss, Goettingen, Germany). Pictures were taken by a cooled CCD digital camera (Retiga 1300, Qimaging, Burnaby, BC, Canada) and visualized with ImageProPlus software (MediaCybernetics, Bethesda, MD).