Cytochrome c d18c7

Dulbecco's modified Eagle's medium (DMEM) (Life Technologies Corporation, Grand Island, NY 14072, USA). The primary antibodies Bax (#2772), Bcl‐2 (124) (#15071), Cytochrome c (D18C7) (#11940) were purchased from Cell Signaling Technology (Danvers, MA, USA). The secondary antibodies anti‐rabbit IgG, HRP‐linked Antibody (#7074) anti‐mouse IgG, HRP‐linked Antibody (#7076), and β‐actin (13E5) (#4967) were purchased from Cell Signaling Technology (Danvers, MA, USA). Ethanol and methanol were purchased from RCI Labscan Limited (Bangkok 10330, Thailand). Folin & Ciocalteu's phenol reagent, 2,4,6‐Tris (2‐pyridyl)‐s‐triazine (TPTZ), 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) and (±)‐6‐Hydroxy‐2,5,7,8‐tetramethylchromane‐2‐carboxylic acid (Tro‐lox), cyanidin‐3‐glucoside (C3G; purity ≥99%) were purchased from Sigma‐Aldrich (St. Louis, MO, USA).

Samples were resolved by SDS-PAGE (7.5–15% gels), transferred to Immobilon-P membranes (Millipore Inc.) and immunoblotted as indicated. Primary antibodies were used to detect Cox IV (3E11, Cell Signaling), cytochrome c (D18C7, Cell Signaling), Drp1 (BD Biosciences; D6C7, Cell Signaling; H-300, Santa Cruz biotechnology), Flag (Sigma), GAPDH (Abcam), GFP (FL, Santa Cruz biotechnology; Roche), GST (GE Healthcare), HA (Sigma), Mff (Proteintech; Sigma), RhoGDI (Abcam), SENP3 (D20A10, Cell Signaling), and β-actin (Sigma). Immune complexes were detected either using HRP-conjugated secondary antibodies (Sigma) followed by enhanced chemiluminescence (Thermo Scientific Pierce) or using fluorescent secondary antibodies (LI-COR). Each immunoblot presented is representative of at least three experiments carried out using different cell populations.

To detect the expression of exosomes associated markers, exosomes pellets and cultured cells were lysed in RIPA lysis buffer containing 1X protease inhibitor. Proteins (50 μg for both exosome and cell lysate) were subjected to 12% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Millipore, Germany). Specific antibodies used for detect exosome associated markers include Alix (3A9) (1:1000, 2171, Cell Signaling Technology, US), CD63 (1:200, sc-5275, Santa Cruz, US), CD81 (1:200, sc-166029, Santa Cruz, US). Cytochrome C (D18C7) (1:1000, 11940, Cell Signaling Technology, US) was used to determine mitochondria contamination.
The size distribution of HUVECs derived exosomes was measured by Nanosight NS300 (Malvern, UK). The morphology of exosomes was visualized by a high-resolution transmission electron microscope (TEM, Hitachi HT7700, Japan). The procedure of exosome staining prepared for TEM was performed as previously described 68 .