Vglut2

EVs used in Western blotting analysis were purified from patient plasma using ExoQuick Ultra (System Biosciences, EQULTRA-20A-1), following the manufacturer’s instruction. Final elutes were dissolved in RIPA buffer (1/10 of elute volume). Signals were detected and quantitated with the LI-COR Odyssey system. To calculate marker ratios, intensities of protein bands of each marker were divided by signals of CD9 bands of the same patient. To rescale the marker ratios, the median value of each marker ratio in the whole patient cohort was set to be 100; then all other values were rescaled accordingly. Antibodies used were CD9, Cell Signaling Technology 13174; VGLUT2, Cell Signaling Technology 71555; GPC4, R&D Systems, Bio-Techne, MAB9195; MUC1, BD Biosciences 555925; EGFR, Cell Signaling Technology 4267; EPCAM, BioLegend 118201; CD44v6, Thermo Fisher Scientific BMS125; CD14, BioLegend 367101; annexin A11, Thermo Fisher Scientific MA5-25052; and sLeX, BD Biosciences 551344.

Protein expression analysis was obtained from four mice from the control group and 4 mice from the ghrelin-treated group. Frozen hypothalamus and cortex were homogenized in protein extraction buffer (30 mM Hepes, pH 7.4, 150 mM NaCl, 10% glycerol, 0.5% sodium deoxycholate [DOC], 1% Triton X-100 with phosphatase and protease inhibitors). Fifty micrograms of protein were analyzed on 10% SDS-PAGE gels and then transferred onto PVDF membranes (Millipore). The following primary antibodies were used: GAD65/GAD2 (1/1000; Cell Signaling ref. 5843), VGLUT2 (1/1000; Cell Signaling ref. 71555), and β-actin (1/50,000; Sigma-Aldrich). Blots were incubated with the appropriate IgG-HRP-conjugated secondary antibody. Protein bands were visualized using the ECL immunoblotting detection system (GE Healthcare) and developed on an ImageQuant LAS4000 mini Fuji luminescence imagining system. The bands were quantified by densitometry using ImageJ analysis software.