Chemidoc xrs imaging system and analysis software

The isolated exosomes were collected and the protein concentration determined by using the BCA protein assay (Pierce, Waltham, MA). Protein samples were subjected to electrophoresis on polyacrylamide gels (Bio-Rad, Hercules, CA) and then transferred to polyvinylidene fluoride membranes (Bio-Rad). Subsequently, membranes were blocked, rinsed, and incubated with primary antibodies against HSP70 (1:1000), CD9 (1:500) and CD63 (1:500), which were used as exosomal markers (abcam, England). After overnight incubation at 4 °C, membranes were washed and incubated with their corresponding secondary antibody conjugated with horseradish peroxidase. Protein bands were detected with an enhanced chemiluminescence detection kit (GE Healthcare, Piscataway, NJ) and visualized through h a chemiDocxrs imaging system and analysis software (Bio-Rad, San Francisco, CA).

We isolated the extracted total proteins through 10% SDS-PAGE, followed by transfer on PVDF membranes (EMD Millipore, Billerica, MA, USA). Thereafter, membranes were subjected to 5% skimmed milk powder blocking in Tris-buffered saline that contained 0.1% Tween-20 (TBS-T) under 37 °C for a period of 30 min, rinsed with TBS-T four times and then probed using primary antibodies under 4 °C overnight. In this assay, the primary antibodies (dilution, 1:1000) used were provided by Abcam (Cambridge, MA, USA), as shown below, anti-β-catenin, anti-GSK-3β, anti-Cyclin D1, anti-N-cadherin, anti-MMP-2, anti-β-actin, anti-LEF1. Later, the membranes were washed extensively and probed using HRP-labeled goat anti-rabbit IgG polyclonal secondary antibody (cat. no. 7074; dilution, 1:2000; CST Biological Reagents Co., Ltd.) under ambient temperature for 1 h. Later, enhanced chemiluminescence (Pierce; Thermo Fisher Scientific, Inc.) was conducted to measure the immunoreactivity, whereas the ChemiDoc XRS imaging system and analysis software (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was utilized for visualization, with GAPDH being an endogenous reference.