Ab117600

Sample protein concentrations were measured using the Pierce^TM BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), and an equal amount of protein (10 µg) of each sample was separated by SDS-PAGE by using NuPAGE Sample Reducing Agent (Thermo Fisher Scientific, Waltham, MA, USA) on precast NuPAGE 4–12% Bis-Tris Protein Gels (Thermo Fisher Scientific, Waltham, MA, USA). Thereafter the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and probed using anti-Calnexin (ab22595, Abcam, Cambridge, UK), anti-Alix (ab117600, Abcam, Cambridge, UK) or Syntenin-1 (TA504796, OriGene, Rockville, MD, USA) antibodies as per manufacturer’s instructions. The secondary antibodies used were goat anti-mouse IRDye 800 CW (926-32210, LI-COR Biosciences, Lincoln, NE, USA) or donkey anti-rabbit IRDye 800 CW (926-32213, LI-COR Biosciences, Lincoln, NE, USA). Precision Plus Protein Dual Color Standards ladder was utilized to determine the size of protein bands (Bio-Rad Laboratories, Hercules, CA, USA). The final blots were imaged using the LI-COR Odyssey CLx infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA).

Unless otherwise noted, all primers and restriction enzymes were purchased from Takara Biotechnology (catalog number Xho I 1635, Sma I 1629). All primary antibodies were purchased from Abcam: Anti-HIV TAT (N3), ab63957; EVs Anti-CD63[EPR5702], ab134045; Anti-ALIX [3A9], ab117600; Anti-Androgen Receptor (AR-V7 specific) [EPR15656], ab198394. The secondary antibody was purchased from Odyssey (Goat anti-Mouse antibody, IRDye 680). Lipofectamine 3000 Transfection Reagent was purchased from Life Technology (L3000008). Plasmid pET-44bC vector was purchased from Novagen (71123–3). T4 DNA ligase was purchased from New England BioLabs (M0202S). His GraviTrap Kit for protein purification was purchased from GE Healthcare (11–0036-90 AA). TRIzol was purchased from Life Technology (15596026). Reverse transcription kit and real-time quantitative PCR kit were purchased from Takara Biotechnology (RR036A and RR820A). Cell Counting Kit-8 (CCK8) was purchased from Dojindo (CK04). Negative control siRNA (siRNA-NC), Cy5-labeled control siRNA (Cy5-siRNA), FAM-labeled control siRNA (FAM-siRNA), therapeutic siRNAs targeting FLOH1, NKX3, DHRS7 genes, and the negative control siRNA were synthesized by RiboBio Co., Ltd. All other reagents and chemicals were of analytical grade and used without further purification.

Tissues, cells, and Exos were lysed using RIPA buffer (KeyGEN BioTECH), followed by protein quantitation using a Bradford Protein Assay kit (KeyGEN BioTECH). Briefly, lysates were subjected to SDS–PAGE and transferred onto PVDF membranes (Millipore). Membranes were incubated overnight at 4° C with primary antibodies specific for the following proteins, as required by each experiment: TSG101 (1:1000 dilution; ab125011, Abcam), CD9 (1:1000 dilution; ab92726, Abcam), ALIX (1:1000 dilution; ab117600, Abcam), IRAK1 (1:1000 dilution; #511166, ZEN BIO), TRAF6 (1:1000 dilution; #380803, ZEN BIO), NFκB (p65) (1:1000 dilution; ab76302, Abcam), GAPDH (1:5000 dilution; 60004-1-Ig, Proteintech), IL-6 (1:1000 dilution; ab6672, Abcam), TNF-α (1:1000 dilution; Ab8348, Abcam), IL-1β (1:1000 dilution; 12242S, CST), and Drosha (1:1000 dilution; ab183732, Abcam). Thereafter, membranes were incubated with the relevant horseradish peroxidase (HRP)-conjugated secondary antibody to visualize protein spots using an enhanced chemiluminescence (ECL) kit (Thermo Scientific). Housekeeping protein GAPDH was selected as an internal control. Each experiment was conducted in triplicate.

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.

Western blotting for the EV markers CD63, CD9, and HSP70 (EXOAB-KIT-1, System Biosciences), TSG101 (ab83, Abcam), and ALIX (ab117600, Abcam) was undertaken as previously described by Lötvall et al. (63 (link)). The purity of EV samples was tested for contaminating intracellular organelles by ATP5A (mitochondria) (15H4C4, Abcam), nuclear fractions by histones (histone H3) (D1H2/4499P, Cell signaling), and platelets by CD41 (ab63983, Abcam). Briefly, protein quantity in EVs was detected by lysing EV pellets in CelLytic MT Cell LysisReagent or RIPPA buffer (Cell Signaling) with added protease inhibitors (Roche Complete). To ensure equal loading, protein concentration was measured by bicinchoninic acid assay (Thermo Scientific), and 10–40 μg protein was loaded per well. SDS page samples were separated on 4%–12% bis-tris gradient gels (Life Technologies) under nonreducing conditions. Separated samples were transferred to PVDF or nitrocellulose membranes, and nonspecific binding was blocked with 5% milk in PBS-Tween. Membranes were incubated with antibodies overnight, and membranes were washed and incubated with a secondary antibody conjugated to horseradish peroxidase. Membranes were washed again before incubation with enhanced chemiluminescence substrate (Pierce ECL, ThermoFisher Scientific) and imaged on a Bio-Rad ChemiDoc MP Imaging system.

Exosomes or whole cell lysates were loaded onto 4–20% gradient SDS-PAGE gels (Bio-Rad) and transferred onto nitrocellulose membranes. The membranes were blocked with Block-One^TM blocking reagent (Nacalai Tesque) and then incubated with primary antibodies using Can Get Signal^TM solution 1 (TOYOBO) overnight at 4 °C and followed by incubation with secondary antibodies conjugated with HRP using Can Get Signal^TM solution 2 (TOYOBO) for 60 minutes at room temperature. The following primary antibodies were used: goat polyclonal anti-adiponectin (AF1119, R&D); goat polyclonal anti-T-cadherin (AF3264, R&D); rabbit monoclonal anti-α-tubulin (11H10, Cell Signaling); rat monoclonal anti-mouse CD63 (clone R5G2, MBL); rabbit polyclonal anti-syntenin (ab19903, abcam); rabbit polyclonal anti-Tsg101 (ab125611, abcam); goat polyclonal anti-MFG-E8 (AF2805, R&D); mouse monoclonal anti-Alix (ab117600, abcam); rabbit polyclonal anti-Nox2/gp91 phox (ab80508, abcam). CD63 was detected under non-reducing conditions. Chemiluminescence signals developed with Chemi-Lumi One Super^TM (Nacalai Tesque) were visualized by ChemiDoc Touch^TM and quantitated using Image Lab software (Bio-Rad).