Chemiluminescent detection reagent

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.

Equal amounts of protein from cells (or EVs) were separated via sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and then transferred to PVDF membranes (Millipore). After blocking in 5% non‐fat milk with Tris‐buffered saline Tween‐20 (TBST, Heart), the membrane strips were incubated with primary antibodies in terms of anti‐β‐actin (1:1000; Proteintech, Rosemont, USA; #60008‐1‐lg), anti‐ALIX (1:1000; Cell Signaling Technology, Danvers, MA, USA; #2171), anti‐CD9 (1:1000; Cell Signaling Technology; #13174), anti‐CD81 (1:1000; Abcam, Cambridge, Britain; ab109201), anti‐VEGF (1:1000; Abcam; ab46154), anti‐Sufu (1:1000; Cell Signaling Technology; #2520) and anti‐Gli1 (1:1000; Cell Signaling Technology; #3538) overnight at 4°C. Then, the membranes were washed using TBST three times and incubated with HRP‐conjugated secondary antibodies (1:5000, Proteintech; SA00001‐1 or SA00001‐2) for 2 h at room temperature. Subsequently, the protein bands were detected with the chemiluminescent detection reagent (Zeta Life) and analysed with ImageJ software. The expression levels of proteins were quantified based on the β‐actin level.