Bradford protein assay

Cell lysates were prepared using RIPA lysis butter (Beyotime P0013) and protein concentrations estimated using a Bradford protein assay (Sangon Biotech, China). Thirty μg of total protein was resolved by SDS-PAGE electrophoresis and then electro-transferred onto PVDF membranes (Bio-Rad, USA). Membranes were blocked with 5% skim milk in Tris-Buffered Saline-Tween-20 (TBST) for 1.5 h before incubating with diluted primary antibodies at 4° C overnight as follows: ASIC1 (Abcam ab240896, 1:1000), ASIC2 (Proteintech 17851-1-AP, 1:1000), ASIC3 (Alomone ASC018AN0702, 1:200), ASIC4 (Proteintech 12003-1-AP, 1:1000), p16 (Santa Cruz sc-166760, 1:500), p21 (Proteintech 10355-1-AP, 1:500), p53 (Proteintech 10442-1-AP, 1:1000), pRb1 (Proteintech 10048-2-Ig, 1:500), MMP3 (Proteintech 6338-1-Ig, 1:500), MMP9 (Proteintech 10375-2-AP, 1:500), Aggrecan (Proteintech 13880-1-AP, 1:500), Collagen II (Proteintech 15943-1-AP, 1:500) and β-actin (Proteintech 66009-1-lg, 1:10000). Antibodies were detected with the corresponding species-specific secondary antibodies at room temperature for 1 h and after washing with TBST, immunoreactive bands were observed by enhanced chemiluminescence (ECL) based detection (Thermo U1291095A). Densitometric analyses were performed using Image J with loading normalization performed against the β-actin control.

At 6 days post-siRNA treatment, soluble proteins were extracted from worms and quantified by using the Bradford protein assay (Sangon Biotech, Shanghai, China). Subsequently, the proteins were separated by SDS-PAGE, electrotransferred onto a PVDF membrane (Whatman International Ltd., Kent, UK), and blocked in 5% skim milk in PBS (pH 7.4) containing 0.1% Tween 20 (Sigma, St Louis, MO, USA) (PBST) overnight at 4 °C. The membrane was incubated with specific mouse anti-rSjVAMP2 serum^{43 (link)} at a dilution of 1:100 or β-actin (Cell Signaling Technology, Boston, MA, USA) diluted 1:1000 in PBST for 1 h at room temperature. After three 5-min washes in PBST, the bound primary antibodies were detected using horseradish peroxidase-conjugated goat anti-mouse IgG (Beyotime Biotechnology, Shanghai, China) diluted 1:2000 for 1 h at room temperature. The membrane-bound proteins were exposed to an X-ray film and detected using the enhanced chemiluminescence detection system (GE Healthcare Life Sciences, Stockholm, Sweden).

The concentrations of purified exosomes were determined using Bradford protein assays (Sangon Biotech, Shanghai, China). Proteins (5 μg) from isolated S. japonicum EVs were separated using precast 4–20% polyacrylamide linear gradient gels (Bio-Rad, Hercules, CA, USA). A pre-stained protein standard (Thermo Scientific, Waltham, MA, USA) was used to track protein migration. After running, gels were stained by silver as previously described46 (link) and scanned using a Bio-Rad Molecular Imager FX system (Bio-Rad).