Anti tsg101

The concentration of total protein in EV samples and cells lysed in RIPA buffer was determined using the NanoOrange^™ protein quantitation kit (#N6666, ThermoFisher Scientific, Eugene, OR, USA) according to the manufacturer’s recommendations using a SpectraMax M5e microplate reader (Molecular Devices, LLC., San Jose, CA, USA). Immunoblotting was performed according to the previously described procedure [25 (link)] with the differences that 5 µg of total protein was applied to SDS-PAGE and proteins were visualized with SuperSignal^™ West Femto Maximum Sensitivity Substrate (#34095, ThermoFisher Scientific, Rockford, IL, USA). The following primary and secondary antibodies and dilutions were used: anti-Alix (#sc-271975, 1:500; Santa Cruz Biotechnology, Dallas, TX, USA), anti-Flotillin-2 (#3436S, 1:1000; Cell Signaling Technology, Topsfield, MA, USA), anti-CD9 (#13174, 1:2000; Cell Signaling Technology), anti-TSG-101 (ab125011, 1:5000; Abcam, Cambridge, UK), anti-PCNA (#sc-7907, 1:500; Santa Cruz Biotechnology), anti-Stomatin (#sc-134554, 1:500; Santa Cruz Biotechnology), anti-mouse goat polyclonal antibodies (#2367, 1:5000; Cell Signaling Technology); and anti-rabbit goat polyclonal antibodies (#29902, 1:80,000; Cell Signaling Technology).

Protein extracts were boiled in RIPA buffer (Beyotime) and separated by sodium dodecyl sulfate–polyacrylamide electrophoresis gel electrophoresis (SDS‐PAGE). The proteins were then transferred to polyvinylidene fluoride membranes (Millipore) and probed with anti‐CD9, anti‐CD63, anti‐ALIX, anti‐TSG101, anti‐EGFR, anti‐FXR1, anti‐TLR‐4, anti‐MMP2, anti‐TGF‐β1, anti‐phospho‐STAT5, anti‐STAT5, anti‐phospho‐ERK1/2, anti‐ERK1/2, anti‐phospho‐AKT, anti‐AKT, anti‐Ki‐67, anti‐PTEN (Abcam), anti‐Bcl‐2, anti‐Bax, anti‐E‐Cadherin, anti‐Vimentin, and anti‐GAPDH antibodies (Cell Signaling Technology). Electrochemiluminescence (Millipore) was applied to determine protein expression levels.

Exosomes and total protein were run on 10% denaturing SDS‐PAGE gels, then transferred to nitrocellulose membranes (BioRad), which were incubated with primary antibodies anti‐GAPDH (1:1000, rabbit monoclonal, Santa Cruz), anti‐CD9 (1:2000, rabbit monoclonal, Abcam), anti‐Rab5 (1:1000, rabbit monoclonal, Abcam), anti‐TSG101 (1:1000, rabbit monoclonal, Abcam), β‐actin (1:1000, mouse polyclonal, CST), CXCR‐4 (1:100, rabbit monoclonal, Abcam), IL‐6 (1:1000, rabbit monoclonal, Abcam), IL‐10 (1:1000, rabbit monoclonal, Abcam), Arg‐1(1:1000, rabbit monoclonal, CST), iNOS (1:1000, rabbit monoclonal, Abcam), STAT3 (1:1000, mouse monoclonal, CST), Phospho‐STAT3 (1:2000, rabbit monoclonal, CST), Bax (1:1000, mouse monoclonal, Abcam), PCNA (1:1000, mouse monoclonal, Abcam) at 4°C overnight. Blots were detected with horseradish peroxidase (HRP)‐conjugated goat anti‐rabbit or rabbit anti‐mouse secondary antibody (Invitrogen) for 1 hour at room temperature. Images were quantified using the Super Signal West Pico or Femto chemiluminescent detection system (Pierce).

Proteins were extracted from platelets or endothelial-derived EVs, separated on 10–12% sodium dodecyl sulfate–polyacrylamide gels, and transferred onto PVDF membranes, which were soaked in Tris-buffered saline–Tween (TBST) solution containing 5% bovine serum albumin (room temperature) to block non-specific binding. Subsequently, the membranes were exposed to the primary antibodies: anti-PDI (Abcam), anti-ERP57 (Abcam), anti-ERP46 (Immunoway, Plano, TX, USA), anti-GRP94 (Abcam), anti-CD63 (Abcam), anti-Calnexin (Abcam), and anti-TSG101 (Abcam). After staining overnight at 4 °C, the membranes were washed three times with TBST and incubated with horseradish peroxidase (HRP)-labeled anti-rabbit (ZSGB-BIO, Beijing, China) or anti-mouse (ZSGB-BIO) secondary antibodies at room temperature for 90 min. After washing thrice more with TBST, an enhanced chemiluminescence (ECL) reagent was added, and images were acquired and analyzed quantitatively using Image J.

Western blotting was carried out as described previously [68 (link)]. Firstly, the total proteins in cell lysates were harvested, and then they were separated using SDS-PAGE gels. Later, the proteins that had been separated were transferred to polyvinylidene difluoride membranes, which were then blocked in the fat free milk (concentration of 5%). Subsequently, the following antibodies were incubated under 4 °C overnight: anti-RUNX2 (#12556, Cell Signaling Technology, 1:1000), anti-TSG101 (#ab125011, Abcam, 1:1000), anti-CD63 (#ab217345, Abcam, 1:1000), anti-Calnexin (#ab213243, Abcam, 1:1000), anti-UCHL3 (#D25E6, Cell Signaling Technology, 1:1000), anti-SMAD1 (#AP20642c, Abcepta, 1:1000), and anti-β-ACTIN (#4976, Cell Signaling Technology, 1:1000). After incubated with horseradish peroxidase‐conjugated secondary antibodies (Shengxing Biological, Nanjing, China) for 1 h, the specific bands were visualized with the enhanced chemiluminescence (Tanon). The relative density was measured using ImageJ software (V1.8.0, National Institutes of Health, USA).

Exosomes or MSCs were lysed with RIPA buffer containing protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany). Extracted protein concentration was determined by BCA assay (Beyotime, Shanghai, China). Equal quantities of protein were loaded and run on 10% SDS-PAGE gels and then transferred to polyvinylidene difluoride (PVDF) membranes. Each membrane was blocked in 5% BSA and subsequently incubated overnight at 4 °C with anti-TSG101 (Abcam, UK) and anti-CD63 (Abcam, UK) antibodies for exosome characterization, anti-HIF-1α antibody (Abcam, UK) for MSCs analysis, and anti-Actin antibody (Beyotime, Shanghai, China) for both. After washing, the membranes were incubated with peroxidase-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies (Invitrogen, Carlsbad, CA, USA). Image analysis and blot quantification were performed with Image Quant LAS 4000 mini biomolecular imager (GE Healthcare, Uppsala, Sweden).

EVs and PC-3 cells (used as a positive control) were lysed in RIPA buffer (150 ml NaCl, 1% Triton X-100, 0.5% Na deoxycholate, 0.1% SDS, 50 ml Tris) and the protein concentration was assessed using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, USA) following manufacturer’s instructions. Thirty micrograms of EV and cell proteins were mixed with Laemmli buffer under reduction conditions, denatured for 5 min at 100 °C and loaded on 10% SDS-PAGE gel. Proteins were electroblotted to nitrocellulose membranes and the membranes were blocked with 10% (w/v) fat-free milk and then incubated with the following primary antibodies: anti-TSG101 (Abcam, # ab125011), Calnexin (Abcam, # ab22595), CD9 (Santa Cruz Biotechnology, # sc-13118) and β-actin (Abcam, # ab8224) in 1:1000 dilution. The blots were washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG F(ab’)2-HRP (1:2000) (Santa Cruz, #sc-3837) or chicken anti-mouse IgG-HRP (1:2000) (Santa Cruz, #sc-2962) secondary antibodies, respectively. Protein expression was visualized using Western Blotting Detection Reagent kit (GE HealthCare Lifesciences, Germany).