Molecular imaging system

Transmission electron microscopy (TEM) The exosome pellets were fixed in 3% (w/v) glutaraldehyde and 2% paraformaldehyde in cacodylate buffer, and loaded to copper grids that coated with formvar. After that, copper grids were washed, contrasted in 2% uranyl acetate, and dried, then examine by TEM (Morgagni 268D, Philips, Holland).
Western blot The specific exosomes surface markers, including CD63 (Abcam, Cambridge, British, Catalog number:ab134045), TSG101(Abcam, Cambridge, British, Catalog number:ab125011), and ALIX(Abcam, Cambridge, British, Catalog number:ab275377) were detected. According to routine protocols, the specific markers antibodies (Santa Cruz Biotechnology, California, America) (1:1000) and secondary antibodies (Cell Signaling Technology, Boston, America. Catalog number: 7074) (1:10,000) were incubated with membranes respectively and proteins were exposed by molecular imaging system (BIO-RAD, California, America).
Zeta sizer Nano tracking analysis Size distribution of serum exosomes was detected by dynamic light scattering (DLS) which was used to measure the particle size and size distribution of molecules or particles. The particle size information can be obtained by measuring the Brownian motion information of particles (Zetasizer Nano ZS90, Malvern,United Kingdom).

Protein homogenates were separated by 10% SDS-PAGE electrophoresis and were then transferred to a PVDF membrane (Millipore, USA). PVDF membrane was blocked with 5% skim milk for 1 h at room temperature (RT). Primary antibody anti-RCAN1 (1:1000, Abcam, USA) and rabbit anti-human GAPDH polyclonal antibody (1:2000, Kangwei, Shanghai, China) were added and incubated at 4°C overnight. The membrane was washed 3 times for 5 min each time with TPBS, and then the secondary antibodies (goat-anti-rabbit, 1:2000, Kangwei, Shanghai, China) were incubated at RT for 1 h. The membrane was washed 3 times with TPBS. The signal was visualized by chemiluminescence and the relative expression levels were analyzed by Bio-Rad molecular imaging system (Hercules, CA).

Glycosylase/lyase assays were performed with 20 nM lesion-containing substrate and increasing concentrations of enzyme in 20 mM HEPES pH 7.5, 150 mM NaCl, 2 mM EDTA with 200 μg/ml BSA at 25°C for 30 mins. The reactions were stopped with the addition of an equal volume of formamide loading buffer (98% formamide, 5 mM EDTA, 0.1% xylene cyanol and 0.1% bromophenol blue) to assay for both glycosylase and lyase activities. The reaction products were separated from the uncleaved substrates with a 12% (w/v) denaturing polyacrylamide gel and quantified with an isotope imaging system (Molecular Imaging System, Bio-Rad).