Anti goat

EVs were isolated from 10 ml of conditioned medium of 3T3-L1 adipocytes treated with either 0.5 mM palmitic acid or control as described above. The pelleted EVs were then resuspended in 50 µl of RIPA buffer (Cell Signaling, Danvers, MA) with cOmplete proteinase inhibitors (Roche). For circulating EVs, the starting volume of whole blood was 900 µl for mouse and 500 µl for human. Circulating EVs were extracted as described above and resuspended with 200 µl of RIPA buffer with proteinase inhibitors. 25 µl of EVs were mixed with β-mercaptoethanol-reduced 6× sodium dodecyl sulfate buffer (Biomiga, San Diego, CA) and resolved by Criterion™ TGX Any kD™ Precast Gel (Bio-Rad, Hercules, CA). Proteins were transferred to 0.2 µm nitrocellulose membrane (Bio-Rad) and blocked for 1 h with 5% bovine serum albumin in TBS, 0.05% Tween 20. Blots were then hybridized overnight using antibody perilipin A (One World Lab, San Diego, CA or ProSci Inc., Poway, CA), cleaved caspase 3 (D175) (Cell signaling), caspase 3 (Cell signaling), phospo-MYPT1 (T696) (Cell signaling), and MYPT1 (Cell signaling). Secondary antibodies used were anti-rabbit (Cell Signaling; 1:2000) and anti-goat (R&D; 1:2000), respectively. Proteins were visualized by SuperSignal West chemiluminescence substrate (Pierce biotechnology, Rockford, IL). Band intensity was analyzed using Image Lab (Bio-Rad).

Western blots for MME were conducted as previously described.^{25 (link)} Primary antibody (Goat anti-Mouse Neprilysin/CD10, product number AF1126, R&D Systems) was used at a dilution of 1:2000 (volume fraction) and the secondary antibody was antigoat (product number HAF017, R&D Systems). Urine proteins were loaded with equal amount of protein per well (3 μg) and a second series of gels were loaded with equal volume of urine (5 μL) per animal. Densitometric analysis was conducted using a GS-800 densitometer with ImageQuant software (Bio-Rad).