Exosome protein extraction kit

Some markers were quantified by western blot. In brief, total proteins were extracted using RIPA Lysis and Extraction Buffer or Exosome Protein Extraction Kit (EZBioscience, Roseville, CA, USA) according to the protocols. Then, proteins were isolated by 10% SDS-PAGE and electro-transferred on PVDF membranes (Bio-Rad, Hercules, CA, USA). Subsequently, the membranes containing proteins were seriatim incubated with the primary and secondary antibodies for indicated time, including anti-heat shock protein 70 (anti-HSP70; ab47454; Abcam, Cambridge, MA, USA), anti-CD63 (ab134045; Abcam), anti-CD81 (ab109201; Abcam), anti-E-cadherin (ab40772; Abcam), anti-N-cadherin (ab76011; Abcam), anti-Vimentin (ab8069; Abcam), anti-ITGA6 (ab235905; Abcam) and anti-GAPDH (ab8245; Abcam), and Goat Anti-Mouse/Rabbit (ab205719 and ab205718; Abcam). The protein signals were visualized using the ECL Western Blotting Substrate (Bio-Rad) and quantified using Image J software (NIH, Bethesda, MA, USA).

Raw264.7 cells and M2 macrophages were lysed using the RIPA lysis buffer on ice. Proteins were extracted from M2-exos using the exosome protein extraction kit (EZBioscience, Roseville). Afterwards, an equal amount of proteins were run to separate using the 10% SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica). The membranes were blocked using 5% skim milk and incubated the following primary antibodies at 4 °C overnight. The antibodies (Abcam, Cambridge) including anti-TSG101 (ab125011, 1/1000), anti-CD9 (ab263019, 1/1000), anti-TNF-α (ab183218, 1/1000), anti-TRAF2 (ab126758, 1/1000), anti-FADD (ab124812, 1/1000), and anti-GAPDH (ab9485, 1/2500). A secondary antibody (ab97051, 1/10,000) was continued to incubate with the membranes at room temperature for 1 h. The bands were visualized using an ECL western blotting substrate kit (Abcam).