Anti alix

Protein extracts from cells were obtained as previously described [61 (link)]. Briefly, proteins samples were extracted by homogenization using a TissueLyser II (QIAGEN, Tokio, Japan) in cold RIPA buffer (200 mM Tris/HCl (pH 7.4), 130 mM NaCl, 10% (v/v) glycerol, 0.1% (v/v) SDS, 1% (v/v) Triton X-100, 10 mM MgCl₂) with anti-proteases and anti-phosphatases (Sigma-Aldrich; St Louis, MO, USA). Twenty-five μg cell lysates and the VEs obtained from the same amount of cells per plate from at least three independent experiments were separated in 10% SDS-PAGE gels and electroblotted onto nitrocellulose membranes as previously described [13 (link)]. Primary anti-PAkt, and anti-Akt were purchased from Cell Signaling Technology (MA, USA); anti- Alix, anti-Ceruloplasmin, anti-TSG101, anti-CD81, anti-Syntenin, and anti-Mimecan from Sta. Cruz Biotechnology (CA, USA); anti-TGFBeta Ig-h3 (NMP2-67186; dilution 1:500) from Novus Biologicals (NovusBio, CO, USA) and anti-Perilipin-1 from Abcam. Data were expressed as percentages corrected towards GADPH (arbitrary units) in Western blots with mean ± SEM. Data analyses were conducted using GraphPad Prism 6 software.

The protein extracts of cells (or EVs, see the “Production of EVs derived from H-DPSCs and P-DPSCs (H-EVs and P-EVs)” section) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Then, the membranes were blocked in 5% nonfat milk in Tris-buffered saline Tween-20 (TBST, Heart). After incubating with primary antibodies including anti-β-actin (1:1000; Proteintech, Rosemont, USA; #60008-1-lg), anti-ALIX (1:1000; Cell Signaling Technology, Danvers, MA, USA; #2171), anti-HSP-70 (1:1000; Cell Signaling Technology; #4876), anti-CD9 (1:1000; Cell Signaling Technology; #13174), anti-CD81 (1:1000; Abcam, Cambridge, Britain; ab109201), anti-VEGF(1:1000; Abcam; ab46154), and anti-AngII (1:100; Santa Cruz, CA, USA; sc-74,403) at 4 °C overnight, the membranes were washed three times with TBST. Then, the cells were incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000, Proteintech; SA00001-1 or SA00001-2) for 2 h at room temperature. Subsequently, blots were detected using chemiluminescent detection reagent (Zeta Life, CA, USA), and the protein bands were analyzed with ImageJ software. β-actin was employed as the housekeeping gene for internal normalization.

Cells were obtained from the American Type Culture Collection (Manassas, VA, USA). All cells were cultured in appropriate medium containing 10% fetal bovine serum (FBS; Welgene, Gyeongsan, Korea), 1% penicillin-streptomycin, and 100 μg/mL gentamycin (Thermo Fisher Scientific, Waltham, MA, USA). All cell lines tested negative for mycoplasma contamination. The lines were authenticated by standard morphologic examination using microscopy. FTY720 was purchased from Echelon Biosciences (Salt Lake City, UT, USA). N-acetyl-l-cysteine was obtained from Calbiochem (San Diego, CA, USA). E64D and NBD-FTY720 were purchased from Cayman Chemical (Ann Arbor, MI, USA). TNF-α and z-VAD-fmk were purchased from R&D Systems (Minneapolis, MN, USA). PD98059, SB203580, SP600125, NecroX-5, and pepstatin A were obtained from Enzo Life Science (Farmington, NY, USA). Anti-PARP, anti-Cathepsin D, anti-LAMP1, and anti-Beclin 1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-ERK, phosho-p38, anti-phospho-JNK, and anti-Alix were purchased from Cell Signaling Technology (Beverly, MA, USA). The anti-ATG7 antibody was obtained from ProSci Inc. (Poway, CA, USA). The cycloheximide, H₂O₂, 3-methyladenine, actinomycin D, glutathione ethyl ester, and anti-actin antibody were obtained from Sigma (St. Louis, MO, USA).