Calnexin

Total proteins were isolated with RIPA lysis buffer (Beyotime) and quantified with a BCA kit. An equal amount of protein lysate from each sample was separated on SDS-polyacrylamide gels, and the protein bands were transferred onto polyvinylidene difluoride membranes that were subsequently blocked with 5 % skimmed dry milk. Next, the membranes were incubated with the following antibodies: anti-HIF-1α (Hypoxia Inducible Factor 1 Subunit Alpha, Abcam, Cambridge, MA, USA), anti-CD63 (Abcam), anti-TSG101 (Tumor Susceptibility 101, Abcam), anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase)anti-Flotillin (Abcam), anti-SATB2 (Abcam); anti-phosphorylated (p)-MEK (Abcam), anti-MEK (Abcam), anti-phosphorylated (p)-ERK (Affinity Biosciences, Cincinnati, OH, USA), anti-ERK (Affinity); anti-E-cadherin (Cell Signaling Technology, Danvers, MA, USA), anti-N-cadherin (Cell Signaling Technology), anti-Vimentin (Cell Signaling Technology) at 4℃ overnight and then incubated with a secondary antibody for 1 h at room temperature. β-actin (Abcam) and Calnexin (Boster, Wuhan, China) were served as a loading control and a negative control for exosomal markers, respectively. The chemiluminescent signals were detected and the immunostaining intensity of each band was quantified using a Bio-Rad ChemiDoc XRS system (Bio-Rad, Hercules, CA, USA).

The proteins were isolated with RIPA lysis buffer containing protease and phosphatase inhibitors, PMSF, separated by 10–15% SDS-PAGE, and transferred to a PVDF membrane (Millipore, USA). Next, the PVDF membranes were blocked with 5% milk for 1 h and incubated overnight at 4°C with primary antibodies against CD9 (1 : 1,000; Cell Signaling Technology), TSG101 (1 : 1,000; Boster), CD81 (1 : 800; Proteintech), calnexin (1 : 1,000; Boster), α-SMA (1 : 1,000; Boster), vimentin (1 : 1,000; Cell Signaling Technology), collagen I (1 : 1,000; Boster), periostin (1 : 1,000; Proteintech), CD31 (1 : 1,000; Arigo), Angptl4 (1 : 800; Cell Signaling Technology), cyclin D1 (1 : 800; Cell Signaling Technology), β-catenin (1 : 800; Cell Signaling Technology), and GAPDH (1 : 2,000; Cell Signaling Technology). The next day, the secondary antibodies (1 : 3,000; Cell Signaling Technology) were incubated with the membranes for 1 h at 37°C. The bands were visualized by enhanced chemiluminescent image analysis.