Novex bolt transfer buffer

Western blot analysis was performed to analyse CD63 (exosome marker) expression. Cell Lysis Buffer (Cell Signaling Technology) was added to MC4 exosomes (MC4exo) in PBS, sonicated and total protein concentration measured via Bio‐Rad Protein Assay Dye Reagent Concentrate. Total protein (10 μg) was resolved by SDS‐PAGE and transferred to a PVDF membrane in a Mini Trans‐Blot Cell (Bio‐Rad Laboratories Inc.) at 100 V for 2 h in Novex BoltTM transfer buffer (Thermo Fisher Scientific). After blocking with 5% skim milk in Tris‐buffered saline with Tween‐20 (TBS‐T) at RT (1 hr), the PVDF membrane was incubated with anti‐CD63 rabbit antibody [EPR21151] (1:5000; Abcam) overnight at 4°C and probed with anti‐rabbit IgG, HRP‐linked antibody (1:2000; Cell Signaling Technology). Chemiluminescent detection was performed using SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) in a ChemiDoc image system (Bio‐Rad Laboratories Inc.).

Western blot analysis was performed to detect the expression levels of ALP, RANKL, and OPG. In brief, the cells were harvested by centrifugation, and then resuspended in PBS. Following sonication for cell lysis, the total protein concentration in the supernatants was measured using Bradford assay. Total protein (5 µg) was resolved by SDS-PAGE under non-reducing conditions, and subsequently transferred to a PVDF membrane in a Mini Trans-Blot Cell (Bio-Rad Laboratories Inc., Hercules, CA, USA) at 100 V for 2 h in Novex BoltTM transfer buffer (Thermo Fisher Scientific). After blocking with 5% skim milk in Tris-buffered saline with Tween 20 (TBS-T) for 1 h at room temperature, the PVDF membrane was incubated with mouse anti-FLAG antibody (1:5000; cat. no. F2555; Sigma-Aldrich; Merck KGaA) for 1 h at room temperature and sequentially probed with horseradish peroxide (HRP)-conjugated rabbit anti-mouse IgG antibody (1:5000; cat. no. A9044; Sigma-Aldrich). The secondary antibody was detected using a tetramethylbenzidine (TMB) substrate reagent according to the manufacturer’s instructions (BD Biosciences, San Jose, CA, USA). The chemiluminescent signals on the PVDF membrane were visualized using a ChemiDoc image system (Bio-Rad Laboratories Inc.).