A phosphatase inhibitor

Western blot analysis of cells, samples and exosome lysates was performed according to the manufacturer's instructions and a previously described protocol 28 (link). Briefly, cells, samples and exosomes were lysed with RIPA buffer (Solarbio, Beijing, China) supplemented with phenylmethylsulfonyl fluoride (PMSF) and a phosphatase inhibitor (Solarbio), and lysates were incubated on ice for 30 min to ensure membrane disruption. Protein concentrations were determined using a bicinchoninic acid (BCA) kit (Beyotime, Shanghai, China) following the manufacturer's recommendations, and electrophoresis was performed using 10% acrylamide gels. The following primary antibodies were used: anti-EGFRvIII, anti-EGFR, and anti-p-AKT Ser473 (Cell Signaling Technology (CST), Boston, MA, USA, 1:1,000 dilution), anti-PTRF (Abcam, Cambridge, MA, USA, 1:1,000 dilution), anti-CD63 (Santa Cruz Biotechnology, CA, USA, 1:500 dilution) and anti-GAPDH (Millipore, Billerica, MA, USA, 1:2,000 dilution). Anti-mouse and anti-rabbit horseradish peroxidase-conjugated secondary antibodies were purchased from Promega (Madison, WI, USA, 1:10,000 dilution). Immunoblots were developed using G:BOX F3 (Syngene, Cambridge, UK).