Ecl kit chemiluminescence reagents

sEVs extracted above were added to 5×SDS gel loading buffer [30 mM Tris-HCl (pH 6.8), 2% SDS, 0.05% bromphenol blue, 12.5% glycerol, and 2.5% mercaptoethanol] and then boiled for 30 min. Then 20 μg of sEVs were added into each lane on 10% SDS polyacrylamide gels and the mixtures were resolved for Western blotting analysis. The samples were blotted onto PVDF membranes (Millipore, Boston, MA, USA), which were blocked with 5% milk for 1 hour at room temperature and then probed with relevant primary antibodies of CD63 (ab193349, Abcam, Cambridge, MA, USA), GM130 (ab52649, Abcam, Cambridge, MA, USA), and Alix (sc53540, Santa Cruz Biotechnology, Shanghai, China) at 4°C overnight. Subsequently, membranes were reacted with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature and observed using the ECL kit chemiluminescence reagents (Millipore, Billerica, MA, USA). Similarly, tissue proteins were isolated from hearts with RIPA lysis buffer and 20 μg of proteins per sample were analyzed using primary antibodies against hepatocyte growth factor (HGF, ab83760, Abcam, Cambridge, MA, USA), vascular endothelial growth factor (VEGF, ab64154, Abcam, Cambridge, MA, USA), and platelet derived growth factor BB (PDGF-BB, ab16829, Abcam, Cambridge, MA, USA). Other procedures were the same as described above.

Western blot was performed as previously described [34 (link)]. Briefly, tissue samples from the ipsilateral cortex of sham/TBI mouse brains were homogenized in RIPA buffer (50 mM Tris-HCl at pH 7.4,150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA) with protease and phosphatase inhibitors, followed by denaturation at 95 °C for 10 min. Then the protein samples were separated by SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). Next, the PVDF membranes were blocked with 5% bovine serum albumin for 1 h and incubated with the primary antibodies overnight, including anti-Mer antibody (1:1000, Abcam, ab184086), anti-p-Mer antibody (1:500, Abcam, ab192649), anti-STAT1 antibody (1:1000, CST, 14994), anti-p-STAT1 antibody (1: 1000, CST, 9167), anti-SOCS-1 (1:1000, Abcam, ab62584), anti-SOCS-3 (1:1000, Abcam, ab16030), and anti-GAPDH (1:5000, Abcam, ab8245). After that, the PVDF membranes were disposed of with the relevant secondary antibodies (1:5000) for 1 h at room temperature and observed using the ECL kit chemiluminescence reagents (Millipore, Billerica, MA, USA). The signals of protein bands were detected with the Chemidoc detection system and quantified using Quantity One software (Bio-Rad).

Cells were lysed in RIPA buffer (50 mM Tris-HCl at pH 7.4,150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA) with protease and phosphatase inhibitors, followed by denaturation at 99 °C for 10 min. Then the protein samples were separated by SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). Next, the PVDF membranes were blocked with 5% bovine serum albumin for 1 h and incubated with the primary antibodies overnight, including rabbit anti-STAT6 (1:1000, CST, 5397), rabbit anti-p-STAT6 (1:1000, CST, 9361), rabbit anti-p-p65 (1: 1000, CST, 3033), rabbit anti-IKKα/β (1:1000, CST, 2697) and rabbit anti-tubulin (1:5000, ProteinTech, 11224-1-AP). After that, the PVDF membranes were disposed of with the relevant secondary antibodies (1:5000) for 1 h at room temperature and observed using the ECL kit chemiluminescence reagents (Millipore, Billerica, MA, USA). The signals of protein bands were detected with the Chemidoc detection system and quantified using Quantity One software (Bio-Rad).