Ab217345

EVs were lysed in a buffer containing 20 mM Tris/HCl pH 7.4, 1% sodium dodecyl sulfate (SDS), and a protease inhibitor (complete mini, Roche, Mannheim, Germany). The protein concentration was assessed using the BCA assay (Thermo Fisher Scientific, Schwerte, Germany). Twenty µg of heat-denatured protein was loaded onto a 10% gel, separated, and transferred onto a 0.2 μm nitrocellulose membrane (Whatman, Dassel, Germany). After blocking for 1 h with 5% non-fat dry milk in Tris-buffered saline with 1% Tween-20 (TBS-T), membranes were incubated overnight with the following primary antibodies: CD63 (1:200, Abcam, Cambridge, UK; ab217345), CD9 (1:200, Abcam, Cambridge, UK; ab92726), GAPDH (1:2000, Hytest, Turku, Finland; 5G4). Thereafter, membranes were incubated with the appropriate HRP conjugated secondary antibodies (1:1000) for 1 h at room temperature (RT). Finally, membranes were washed with TBS-T and incubated with an ECL substrate (Thermo Fisher Scientific, Schwerte, Germany). The proteins were visualized using the MF-ChemiBIS 3.2 bioimaging system (Biostep, Burkhardtsdorf, Germany).

Western blots were performed as described (Yang et al., 2019 (link); Yang et al., 2022 (link)) using the following antibodies against IDO1 (ab211017, Abcam), Flag (ab205606, Abcam), CD63 (ab217345, Abcam), CD81 (ab109201, Abcam), RUNX2 (ab23981, Abcam), Calnexin (#2433, CST) and GAPDH (AF2823, Beyotime). The whole protein was extracted by RIPA Lysis Buffer (Beyotime Biotechnology, China), and the concentration was detected by a BCA protein assay kit (Beyotime Biotechnology, China). Cell lysates were kept on ice for 30 min and centrifuged at 16,000 g for 3–5 min at 4°C. Supernatants were collected and boiled in SDS loading buffer, and the same amounts of protein were separated by 10% SDS-PAGE and blotted onto polyvinylidene fluoride membranes (Millipore, United States of America). Bands were visualized using chemiluminescence.

A RIPA buffer containing a protease inhibitor (1×) was used to lyse the EV pellets (harvested as described above) at 95 °C for 5 min. Then, equal amounts of protein (20 μg) were resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel, following which the protein bands were electrotransferred to a polyvinylidene difluoride membrane at 4 °C using a voltage of 100 V, a constant current of 0.1 A, and a run time of 1 h 20 min. Next, the membrane was incubated with 3% skim milk in Tween-containing Tris-buffered saline (TBS-T: 10 mM Tris, pH 8.0, 150 mM, and NaCl solution containing 0.05% Tween 20). Then, after three washes with TBS-T (10 min each), the membrane was incubated overnight at 4 °C with primary antibodies (diluted 1:1000 in 3% skim milk) targeting the following proteins: CD63 (ab217345, Abcam, Cambridge, UK), tumor susceptibility gene 101 protein (TSG101; ab30871, Abcam), and ALG-2-interacting protein X (Alix; ab186429, Abcam). Thereafter, the membrane was washed three times with TBS-T as described above and then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG H&L (1:2000 dilution; ab205718, Abcam) for 1 h, at ambient temperature. Finally, after 5 min of signal development, the bands were visualized using an enhanced chemiluminescence detection system (Son et al., 2017 (link)).

Total proteins from the exosomes or cells were extracted and detected using the bicinchoninic acid kit. Briefly, 40 µg of total protein was separated using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (120 V, 90 min) and transferred to polyvinylidene fluoride (PVDF) membranes (90 V, 90 min). For blocking, 5% non-fat milk was added to the PVDF membranes for 1 h. Anti-CD9 (1: 1,000, ab307085, Abcam, Cambridge, MA, USA), anti-CD63 (1: 1,000, ab217345, ab68418), anti-CD81 (1: 1,000, ab109201), anti-SIRT3 (1: 500, ab189860), anti-AMPK (1: 1,000, ab207442), anti-p-AMPK (1: 1,000, ab133448), anti-LC3II/I (1: 1,000, ab48394), anti-P62 (1: 1,000, ab240635), anti-NLRP3 (1: 1,000, ab263899), anti-apoptosis-associated speck-like protein containing a CARD domain (ASC) (1: 1,000, ab70627), anti-IL-1β (1: 1,000, ab283822), anti-iNOS (1:1,000, ab178945), anti-IL-6 (1: 1,000, ab233706), anti-TNF-α (1:1,000, ab183218), anti-Calnexin (1:1000, ab22595) and anti-GAPDH (1:1,000, ab8245) were added and incubated overnight at 4 °C. Moreover, the horseradish peroxidase-labeled goat-anti-rabbit secondary antibody (1:5,000 diluted) was incubated at 25 ℃ for 2 h. Protein blots were detected using Pierce™ ECL (Thermo Fisher, Waltham, USA) in ChemiDoc MP (Bio-Rad, California, USA).

Mammalian cells cultured near confluence in T75 flasks were washed twice with cold PBS and scraped into 200 µl of Pierce 1x RIPA buffer (Thermo Scientific), supplemented with protease inhibitor cocktail (Millipore Sigma), and then transferred to 1.5 ml microtube kept on ice. Cells were vortexed briefly and disrupted by trituration 10× with a 23 G ¼ inch syringe needle (Becton, Dickinson and Co. Thermo Scientific). To remove unbroken cells and cellular debris, lysates were spun at 17,000 × g for 10 min and the supernatant was collected for downstream applications, as well as for determination of protein concentration by the BCA Protein Assay Kit (Thermo Scientific).
For running Western blots, 30 µg of cell lysates in Sample Buffer was loaded per each well of 4–20% Precast Protein Gel (BioRad, Hercules, CA) and then transferred to PVDF membrane. For blocking, 3% BSA (Sigma-Aldrich) in TBS buffer was used, while antibodies were diluted in 1% BSA in TBS-T. For detection of CD63 protein, we used either anti-mouse CD63 (ab217345) or anti-human CD63 (ab134045) (Abcam, Waltham, MA); for detection of NHE1, the Anti-Na+/H+ Exchanger 1 (extracellular) was used from Alomone Labs (ANX-010, Jerusalem, Isreal); for secondary antibodies, the HRP-conjugated goat anti-rabbit IgG H&L (ab6721, Abcam) was used; primary antibodies were used at 1:2000 and secondary antibodies at 1:10,000 dilutions.