Ab8219

Protein from each sucrose gradient fraction (10 in total) obtained after exosome isolation were separated by SDS polyacrylamide gel electrophoresis[7] (link), [30] (link) using a XCell SureLock electrophoresis system (Life Technologies), transferred to Immobilon-FL polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) and probed with primary mouse monoclonal anti-CD63 (1∶2,000 ab8219, Abcam, Sapphire Bioscience Pty Ltd, NSW, Australia), anti-CD81 (1∶1,500 MAB6435, Abnova, Tapei City, Taiwan) and anti-CD9 (1∶1,500 ab2215, Abcam, Sapphire Bioscience) as previously described exosome enriched markers. [7] (link), [18] (link), [29] , [30] (link) Membranes were washed in Tris buffer saline (pH 7.6) and incubated (1 h) in TBST/0.2% BSA containing horseradish peroxidase–conjugated goat anti-mouse antibody. Proteins were detected by enhanced chemiluminescence with using a SRX-101A Tabletop Processor (Konica Minolta, Ramsey, NJ, USA). The exosomal fractions (from 1.126 to 1.187 g/ml) were pooled (defined as exosomes) and placental alkaline phosphatase (PLAP) protein abundance was determined by Western blot using primary polyclonal antibody anti-PLAP (1∶1,000 ab96588, Abcam, Sapphire Bioscience) using a GS-800 Calibrated Densitometer (Bio-Rad Laboratories).

Protein extracts (40 µl) were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes. After blocking for 1 h with 10% Bovine Serum Albumin in Tween-Tris Buffered Saline 1X (T-TBS), membranes were incubated with primary antibody against CD63 (Abcam, ab8219, 1 µg/ml) O/N at 4 °C. After washing, membranes were incubated with a Peroxidase Horse anti- Mouse IgG, (1:2000, PI-2000, Vector) for 1 h at room temperature (RT). Detection was performed using ECL™ reagents according to the manufacturer’s guidelines (GE Healthcare).

EVs were lysed, denatured in SDS sample buffer (0.4% SDS, 0.2M Tris-HCl, pH 6.8, 5% glycerol, 0.02% bromophenol blue, 1% betamercaptoethanol) at 95°C for 10 mins and resolved in 10% SDS PAGE. Details of western blotting procedures were described elsewhere [26 (link)]. Expression of TSG101 (sc-7964, Santa Cruz TX, USA), CD63 (ab8219, Abcam, Cambridge, UK) and HSP-70 (YM3042, ImmunoWay. TX, USA) were detected by specific antibodies. The quantities of protein in the EV preparations were determined by the Pierce BCA Protein Assay kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. EV-to-protein ratio was determined by the quotient of the number of EVs/ml and the protein concentration.

The primary antibodies used are: rabbit anti-SCAMP5 (ab3432, Abcam;1:500 for WB); rabbit anti-LC3B (L7543, Sigma-Aldrich; 1:3000 for WB); mouse anti-p62/SQSTM1 (ab56416, Abcam; 1:2000 for WB); rabbit anti-TFEB (13372-1-AP, Proteintech; 1:1000 for WB); mouse anti-α-synuclein (610787, BD Biosciences; 1:1000 for WB); rabbit anti-huntingtin (5656P, Cell signaling; 1:1000 for WB); mouse anti-GM130 (610822, BD Biosciences; 1:300 for IF); mouse anti-Giantin (ab37266, Abcam; 1:300 for IF); rabbit anti-ERGIC53 (sc-66880, Santa Cruz; 1:100 for IF); rabbit anti-Calnexin (ab22595, Abcam; 1:2000 for WB); mouse anti-CD63 (ab8219, Abcam; 1:1000 for WB); mouse anti-DYKDDDDK-Tag (FLAG) (M20008, Abmart; 1:3000 for WB; 1:300 for IF; 1:200 for IP); goat anti-DYKDDDDK-Tag (FLAG) (NB600-344, NOVUS; 1:100 for IF); mouse anti-HA-Tag (M20003, Abmart; 1:200 for IP); mouse anti-GFP-Tag (M20004, Abmart; 1:2000 for WB); mouse anti-β-tubulin (M20005, Abmart; 1:3000 for WB); mouse anti-β-actin (M30002, Abmart; 1:2000 for WB).
The drugs used are: Rapamycin (553210, Merck, 100nM); Bafilomycin A1 (196000, Merck, 10-50nM); MG132 (474790, Merck, 3-5μM); Cycloheximide (C7698, sigma, 100μg/ml); Tetanus toxic (T3194. Sigma-Aldrich, 2nM); BrefeldinA (S1536, Beyotime, 2μM).