To prepare cell membrane proteins, vascular endothelial cells were collected via centrifugation at 500 × g for 5 min, followed by washing with PBS. The cell pellet was then lysed using the Mem-PER Plus Membrane Protein Extraction Kit, according to the manufacturer’s instructions (Thermo Fisher Scientific). The extracts were stored at -80℃ until use.
For western blot analysis, total protein was extracted from the exosomes and HUVECs using RIPA lysis buffer and assessed as previously described [18 (link)]. For quantitative protein analysis, exosomes were labeled with iTRAQ reagents using an iTRAQ multiplex kit (AB Sciex, USA) and analyzed as previously described [19 (link)]. Labeled samples were separated and automatically spotted onto a MALDI plate; mass spectra were then acquired using an AB Sciex TOF/TOF 5800 system. All tandem mass spectrometry data were analyzed using MASCOT and Protein Pilot software (version 4.5; AB Sciex) to identify and quantify the corresponding proteins in different groups (Table S2). Protein identification was considered correct based on the selection criteria [19 (link)].
For western blot analysis, total protein was extracted from the exosomes and HUVECs using RIPA lysis buffer and assessed as previously described [18 (link)]. For quantitative protein analysis, exosomes were labeled with iTRAQ reagents using an iTRAQ multiplex kit (AB Sciex, USA) and analyzed as previously described [19 (link)]. Labeled samples were separated and automatically spotted onto a MALDI plate; mass spectra were then acquired using an AB Sciex TOF/TOF 5800 system. All tandem mass spectrometry data were analyzed using MASCOT and Protein Pilot software (version 4.5; AB Sciex) to identify and quantify the corresponding proteins in different groups (Table S2). Protein identification was considered correct based on the selection criteria [19 (link)].
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