Flotillin

Brain tissues, treated cells or EVs were lysed using the Mammalian Cell Lysis kit (Sigma‐Aldrich), as described previously (Liao et al., 2016). Equal amounts of the proteins were electrophoresed in an SDS‐polyacrylamide gel under reducing conditions followed by transfering to PVDF membranes. Blots were blocked with 5% BSA in TBS‐Tween 20. The western blots were then probed with antibodies specific for Iba‐1 (1:1000; 019–19741; Wako), Tsg101 (1:1,000; ab125011; Abcam, Cambridge, MA, USA), Alix (1:1,000, ab117600; Abcam), CD63 (1:1,000; ab216130; Abcam), Flotillin (1:200, Cell Signaling Technology), Calnexin (1:1500; C7617; Sigma‐Aldrich), NF‐κB p65 (1:2,000; ab16502; Abcam), Histone H3 (1:1,000; 9715S; Cell Signaling Technology) and β‐actin (1:5,000; A5316; Sigma‐Aldrich). Secondary antibodies were alkaline phosphatase conjugated to goat anti‐mouse/rabbit IgG (1:10,000; Jackson ImmunoResearch Labs). Signals were detected by SuperSignal West Dura Extended Duration or Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA). All experiments had at least four biological replicates, and representative blots are presented in the figures.

A total of 25 µg protein from human MSC-EV fractions isolated by filtration/ultracentrifugation was resolved by SDS-PAGE under reducing conditions (4–20%; Invitrogen Novex Wedge Well, 1.0 mM; Tris-Glycine, Mini Protein Gel, 10-well; XCell SureLock Mini). The gel was transferred to a polyvinylidene difluoride membrane (0.45 µm; 10 × 10 cm, Thermofisher), which was blocked in tris-buffered saline with 5% bovine serum albumin, then cut based on molecular weight. Sections were incubated with respective primary antibodies overnight: HSP90 (Cell Signaling: 4877S) was used as a positive loading control; Flotillin (Cell Signaling: 3436S) was used as a positive EV marker expected to be detected in all lysates (WCL, MV, EX); IκBα (Cell Signaling: 4814S) was used as cellular marker to screen for cellular contaminants in conditioned media fractions; ATP5A1 (Thermofisher:459240) and COXIV (Cell Signaling: 4850S) were used to probe for mitochondrial proteins. The membrane sections were rinsed three times with tris-buffered saline and incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature with gentle rocking. All primary and secondary antibodies were used at 1:1,000 dilutions. Proteins were detected using enhanced chemiluminescence reagents and a chemiluminescent imaging device (Bio-Rad Chemidoc XRS).

Briefly, cells and EVs were lysed in RIPA buffer supplemented with protease inhibitors (Roche). The protein concentration was determined using a protein assay kit (ThermoFisher Scientific, Waltham, MA, USA) and read in a Qubit 3.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA). To characterize EVs markers, 30 μg of total protein from EVs and from the respective cells were resolved on 12% SDS PAGE gel by PAGE and transferred to Nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). Membranes were incubated with one of the following primary antibodies: Alix, flotillin, CD9, Calnexin, GM130 (All from Cell Signaling; 1:2000) or Cx46 (Santa Cruz Biotechnology; 1:500). All secondary antibodies were horse-radish protein (HRP) conjugated (Abcam). Protein bands were detected using Immobilon Forte western HRP substrate (Millipore, Burlington, MA, USA) and visualized with LI-COR C-Digit Chemiluminescense Western Blot Scanner systems (LI-COR, Inc, Lincoln, USA).

Antibodies for EV markers of CD9 (CellSignalling, 13174, 1:250), Flotillin (CellSignalling, 18634, 1:1000) and GM130 (CellSignalling, 12480, 1:1000) were tested using Western Blotting, using anti-rabbit secondary antibody (CellSignalling, 7074, 1:2000). EVs were separated by 4–20% Gradient Sodium Dodecyl Sulphate-Polyacrylamide Gel (Biorad, 4–20% Mini-PROTEAN® TGX^TM, USA) Gel and Electrophoresis (SDS-PAGE) were applied. Proteins were transferred onto PVDF membrane (Bio-Rad #162-0177, USA) by Pierce G2 fast blotter. Membranes were blocked with 5% skim milk powder, for 1 hour and 30 minutes at room temperature. After the overnight incubation with primary antibody at 4 °C. Membrane was visualized with ChemidocTM XRS + system. Image Lab Software 6.0.1 was used for quantification of images.

Equal amount of protein in an equal volume of each sample was mixed with Laemmli’s buffer and separated by SDS-PAGE. Proteins were transferred to nitrocellulose membranes overnight at 4°C. Following transfer, membranes were blocked with 5% (w/v) milk in PBS-T. Membranes were incubated overnight with primary antibodies: CD63 [1:1000] (EXOAB-CD63A, System Bioscience, Palo Alto, CA), PLAP [1:750] (Abcam, Cambridge, MA), Calnexin [1:1000] and Flotillin [1:750] (Cell Signaling Technology Danvers, MA). HepG2 cell (ATCC) lysate was used as the–ve control for the PLAP antibody and +ve control for the Calnexin antibody. Membranes were then incubated with the appropriate peroxidase labeled secondary antibodies. Protein bands were visualized with chemiluminescence using the ChemiDoc Imaging System (Bio-Rad Laboratories, Hercules, CA).