Mouse secondary antibody

The total protein of the three groups were extracted using RIPA buffer (Beijing Kangwei Reagent Co., Ltd., Beijing, China). Then, proteins were quantified, isolated and transferred onto the PVDF membranes (Roche, Basel, Switzerland). The primary antibody of PLK1 (mouse-anti-human, Millipore Corp, Billerica, MA, USA) was diluted at 1:1,000, then uniformly dropped on the membrane, and left to incubate overnight at 4 °C. After washing the membrane, a mouse secondary antibody (1:1,000, Sigma, USA) was incubated at room temperature for 1.5 h and then visualized using the Kangwei Colorimetric Kit (Beijing, China). At the same time, β-actin was established as an internal reference. Each sample was performed in triplicate.

The BCA protein assay was used for ensuring that equal concentrations of exosomes were prepared for SDS–polyacrylamide gel electrophoresis (SDS-PAGE). After SDS-PAGE, samples were transferred to nitrocellulose membranes. The blots were incubated overnight at 4°C with the following primary antibodies: VSV-G (clone: P5D4), Alix (clone: H270), CD63 (clone: MEM-259), Tsg101 (clone: 4A10), CD81 (clone: sc-166029), GFP (ab 290, ChIP grade), and LDLR (clone: EP1553Y). Next, the membranes were probed with horseradish peroxidase–conjugated anti-rabbit (Sigma-Aldrich) or mouse secondary antibody (Sigma-Aldrich). Last, the blots were visualized with enhanced chemiluminescence reagents (Bio-Rad).

Protein synthesis was measured using the SUrface SEnsing of Translation (SUnSET) method [39 (link)] which measures the incorporation of puromycin into nascent peptide chains. At 24 h after transfection, N2a cells were incubated in puromycin treatment media (1 μg/mL puromycin in DMEM (10% FBS, 1% P/S)). After a 30-min incubation at 37°C, puromycin treatment media was removed and cells were incubated with DMEM (10% FBS, 1% P/S) for 15 min. Cells were then washed with PBS two times and harvested for cell lysis. 20 μg of protein was separated on a 10% SDS-PAGE gel. Proteins were transferred to PVDF membrane using a Trans-Blot Turbo Transfer System (25 V, 1.3 A constant for 20 min; BioRad) and subsequently incubated in TBST buffer containing 5% BSA. Membranes were then incubated overnight with a monoclonal puromycin antibody (clone 12D10, EMD Millipore, Temecula, CA, United States, diluted 1:5000 in TBST and 5% BSA w/v), then washed three times with TBST and 1% BSA for 10 min each. Membranes were incubated for 2 h in TBST containing 3% skim milk plus a secondary mouse antibody (Sigma-Aldrich, 1:5000) then washed three times with TBST for 15 min each. Membranes were visualized on BioRad ChemiDoc imaging system. The total lane density was analysed using Image J [40 (link)].