X ray film

Western blotting was performed in accordance with a previously published method (Chen et al., 2019 (link)). Total proteins were extracted by RIPA lysis buffer with proteinase inhibitor (Roche, Switzerland). In total, 30 μg aliquots of protein were separated in 10% SDS-PAGE gels and then transferred onto PVDF membranes (Millipore, United States). After 1.5 h of blocking with 5% w/v nonfat dry milk buffer, the membrane was incubated overnight with primary antibodies against GM130 (#66662-1-Ig, Proteintech), GAPDH (#10494-1-AP, Proteintech), CD63 (#PA5-92370; Invitrogen), CD9 (#ab92726, Abcam), CD81 (#ab109201, Abcam), S100ß (#ab52642; Abcam), nerve growth factor receptor (NGFR, #8238, Cell Signaling Technology), myelin protein zero (MPZ, #ab31851, Abcam), glial fibrillary acidic protein (GFAP, #3670, Cell Signaling Technology), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta (PIK3CD, #ab109006, Abcam) and phosphorylated serine/threonine kinase (p-AKT, #4060, Cell Signaling Technology). Then, the membrane was incubated with secondary antibodies (Aspen, China) and exposed to X-ray film (UVP, United States).

Total proteins were extracted by RIPA lysis buffer with proteinase inhibitor (Roche, Switzerland). An equal amount of total protein (20–40 μg) was separated by SDS-PAGE (Beyotime Biotechnology, Shanghai, China), transferred into the PVDF membrane (Millipore, USA), and then incubated overnight with primary antibodies specific for HIF1AN (Abcame, USA), β-tubulin (Proteintech, China), CD9 (Abcame, USA), CD81 (Abcame, USA), HSP70 (Abcame, USA), TSG101 (ABclonal, China). Then, the membrane was incubated with secondary antibodies (Aspen, China) for 1 h and exposed to X-ray film (UVP, USA).