Hrp conjugated anti rabbit igg 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).

MBMSCs and IBMSCs were cultured in adipogenic induction medium for the indicated times and lysed in RIPA lysis buffer (Thermo Fisher Scienti c) containing protease and phosphatase inhibitor cocktails (Sigma-Aldrich). The extracted total protein was separated by polyacrylamide gel electrophoresis on 10% gels, and proteins were transferred to a polyvinylidene uoride membrane (Bio-Rad). The membrane was blocked with 5% nonfat milk (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) solution for 1 h. The membrane was then incubated with anti-C/EBPβ (Abcam, Cambridge, UK), anti-C/EBPδ, anti-Ebf-1, anti-KLF5 (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA), anti-PPARγ, anti-C/EBPα, anti-β-actin (Cell Signaling Technology, Danvers, MA, USA), and anti-Zfp423 (Novous Biologicals, Centennial, CO, USA) antibodies overnight at 4°C. After washing with PBS containing Tween-20 (PBS-T), the membrane was incubated with HRP-conjugated anti-rabbit IgG antibody (GE Healthcare, Buckinghamshire, UK) at room temperature for 1 h. Enhanced chemiluminescence (ECL) western blot detection reagent (GE Healthcare, Buckinghamshire, UK) was used to visualize the target protein bands. Protein bands were visualized using the ChemiDoc Imaging System (Bio-Rad). β-Actin protein signals were used as an internal control to normalize protein expression.

Anti-EWS (G-5) antibody was purchased from Santa Cruz Biotechnology. Anti-FUS (SAB4200455) and anti-TAF15 (SAB2102361) antibodies were purchased from Sigma-Aldrich. Horseradish peroxidase (HRP)-conjugated anti-mouse IgG and HRP-conjugated anti-rabbit IgG antibodies were obtained from GE Healthcare. All-trans-retinoic acid (RA) was obtained from Wako Pure Chemicals. Thiamet G was obtained from Cayman Chemical.