Mouse anti human alix

The samples from RBC-derived exosomes, BSA-PEG-micelles, and the peak fractions for the BSA-PEG-lipid-exosomes from size-exclusion chromatography were analyzed by Western blotting. The protein concentrations of all samples were estimated using the BCA assay kit (KeyGEN Biotech). Then, equal amounts of protein were separated on a 10% SDS-PAGE and transferred to the PVDF membrane (Millipore) using a high quality wet protein transfer system (L00686C eBLOT L1, Genscript, Nanjing, China). The membranes were then blocked with 5% skim milk powder in TBST buffer for 1 h at RT. The blots were then incubated overnight at 4℃ with the primary antibodies including mouse anti-BSA (1:2000, Proteintech), mouse anti-human CD63 (1:1000, Santa Cruz), mouse anti-human CD9 (1:1000, Proteintech), or mouse anti-human Alix (1:1000, Santa Cruz). Subsequently, the blots were incubated with HRP-conjugated goat anti-mouse IgG antibodies (1:5000, Cell signaling) for 1 h at RT. Then, the blots were developed using the Enhanced chemiluminescence detection kit (BL520A, Biosharp).