Nb100 1965

Total liver lysates and nuclear or microsome extracts of liver samples were used for Western blotting. The antibody against tubulin (cat. #ab4074) was purchased from Abcam. Antibodies against CYP7A1 (cat. #TA351400) or CYP8B1 (cat. #TA313734) were purchased from Origene. Antibodies against acetyl-CoA carboxylase (ACC) (cat. #3662), cleaved caspase 3 (cat. #9661), total caspase 3 (cat. #9662), phospho-Smad2/3 (cat. #8828), or total Smad2/3 (cat. #5678) were purchased from Cell Signaling Technology (Boston, MA, USA). Antibodies against histone (cat. #sc-393358) or SREBP-1 (cat. #SC-8984) were purchased from Santa Cruz Biotechnology. The antibody against calnexin (NB100-1965) was purchased from Novus.

To identify the exosome markers, the exosomes were extracted with RIPA lysis buffer (89900; Thermo Fisher Scientific) containing a protease inhibitor (87786; Thermo Fisher Scientific). The exosome lysates were mixed with LDS Sample Buffer (B0007; Thermo Fisher Scientific) and boiled at 95°C for 5 minutes. Then, 25 μg of protein was electrophoresed on 4%–12% Bis-Tris Plus gels (NP0335; Thermo Fisher Scientific) with MOPS SDS running buffer (B0001, NuPAGE; Thermo Fisher Scientific). The polyvinylidene fluoride membranes (LC2002; Thermo Fisher Scientific) were blocked with 5% skimmed milk for 1 hour at room temperature. The cells were then incubated overnight with primary antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH, NB100-56875; Novus Biologicals), calnexin (NB100-1965; Novus Biologicals), CD9 (NBP1-28363; Novus Biologicals), CD81 (NBP1-44861; Novus Biologicals), flotillin 2 (NBP1-30881; Novus Biologicals), and TSG101 (NB200-112; Novus Biologicals) at 4°C. The cells were then incubated with the horseradish peroxidase-conjugated secondary antibodies, anti-mouse immunoglobulin G (IgG, 31430; Thermo Fisher Scientific), and anti-rabbit IgG (31460; Thermo Fisher Scientific). Western blot development was performed using ECL (enhanced chemiluminescence) solution (2332638, EzWest Lumi plus; ATTO, Tokyo, Japan).