Protease inhibitor cocktail

The total protein in the cell or sEVs was extracted with the RIPA lysis buffer by using the Protease Inhibitor Cocktail (Transgen, China). The protein concentration was quantified using the BCA protein assay kit, and the protein was subjected to denaturation in boiling water for 10 min. Then, 20 μg of the protein was loaded and separated by 10% SDS‐PAGE and then transferred onto a 0.45‐μm PVDF membrane (Mllipore, USA), followed by blocking in 5% non‐fat milk for 1 h at room temperature. The membrane was incubated with the antibodies to LRP1, Alix, CD63, CD9, HA, EGFP, Calnexin and β‐actin at 4°C overnight. The membrane was washed with TBST thrice, after which the membrane was incubated with HRP‐conjugated secondary antibody at room temperature for 2 h. The protein bands were visualised using the ECL kit(Vazyme, China) and LAS4000 imaging system (Fuji Film, Japan).

The TA muscles were lysed in RIPA lysis buffer with protease inhibitor cocktail and ethylenediaminetetraacetic acid (EDTA) (TransGen, China). Protein concentration was determined using a BCA Protein Assay (TransGen, China). Equal amounts of protein were loaded onto a 10% polyacrylamide gel, then separated and transferred to nitrocellulose membranes (Merck Millipore, Germany). Blots were blocked with 8% non-fat dry milk in Tris-buffered saline and Tween 20 (TBST) for 2 h at room temperature and then incubated with primary antibodies in blocking solution overnight at 4 °C. The primary antibodies included TMEM30A (rabbit polyclonal, ab217330, Abcam, USA) and GAPDH (mouse monoclonal, 60004-1-Ig, Proteintech, USA). Primary antibodies were detected with either an anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibodies (Bio-Rad Laboratories, USA), and signals were developed using a SuperSignal West Pico Chemiluminescent Substrate (Pierce Protein Biology, USA). ImageJ (v1.8.0) was used to calculate the relative density of the protein.

The cells were harvested and processed with RIPA lysis buffer (CST Biological Reagents Co., Ltd.) supplemented with phenylmethylsulphonyl fluoride (Thermo Fisher Scientific, Inc.), protease inhibitor cocktail (TransGen Biotech Co., Ltd.) and phosphatase inhibitor cocktail (TransGen Biotech Co., Ltd.). Western blotting was performed as described below, and the protein samples (20 µg) were separated on 10% gels using SDS-PAGE, and then electro-transferred onto PVDF membranes (Immobilon-P; MilliporeSigma). After blocking with 5% bovine serum albumin (MilliporeSigma) in Tris-buffered saline containing 0.05% Tween 20 for 1 h at room temperature, the PVDF membranes were incubated overnight with the primary antibodies at 4˚C. Following incubation with the corresponding secondary antibodies for 1 h at room temperature, protein chemiluminescence was detected using the BeyoECL Plus kit (Beyotime Institute of Biotechnology) in a KETA GL Imaging System (Wealtec Corp.), and the gray value of the band was quantified using ImageJ (version 1.51; National Institutes of Health). β-actin was used as the control.

Cells were lysed with ice-cold radio immunoprecipitation assay (RIPA) buffer (TRANSGEN Biotech, Haidian, Beijing, China) containing protease inhibitor cocktail (TRANSGEN Biotech, Haidian, Beijing, China). Proteins which was concentrated by a BCA Kit (Pierce, Hangzhou, Zhejiang, China) in the lysates were separated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes. The 5% (wt/vol) nonfat milk was used for membranes block. Subsequently, the membranes were incubated with primary antibodies specific for ZEB1 (1:600; Abcam, Cambridge, MA, USA), GAPDH (1:2000; Yunshan Technology, Pudong, Shanghai, China), Jag1 (1:700; CST, Danvers, MA, USA), Maml2 (1:600; Abcam, Cambridge, MA, USA) or Hey1 (1:700; CST, Danvers, MA, USA). PBS containing 0.05% Tween was used to wash the membranes which further used for the incubation of the appropriate secondary antibodies. The levels of proteins were visualized using ECL reagents (Beyotime, Pudong, Shanghai, China) and imaged by ECL System (GE Healthcare, Milan, Italy). ImageJ software was applied to analyze the optical density of the protein bands.