West pico plus chemiluminescent substrate kit

The samples were denatured and loaded into 8–12% SDS-polyacrylamide gel. The proteins were then transferred onto polyvinylidene fluoride (PVDF) membranes, blocked with milk at 25 °C for 1 h, and incubated with primary antibodies: CD61 (ab119992; 1:1000 dilution), CD14 (ab221678; 1:1000 dilution), and Melan-A (ab210546; 1:1000 dilution; all from Abcam) at 4 °C overnight. The PVDF membranes were further incubated with horseradish peroxidase-conjugated secondary antibody (Thermo Fisher Scientific) and the blots were developed by using a West Pico PLUS Chemiluminescent Substrate kit (Thermo Fisher Scientific).

For SDS-PAGE, neutrophil lysates and N-NVs were added into the protein extraction buffer, and the protein contents were measured with a bicinchoninic acid kit (Sigma-Aldrich, USA). The samples were heated at 95°C for 5 min, and 20 μg of each sample was loaded into a 10% SDS–polyacrylamide gel. The samples were run at 120 V for 2 hours, and the gel was stained with Coomassie blue for 4 hours and then decolorated overnight before the observation. For Western blotting, the protein samples obtained from R-NVs and N-NVs were denatured and loaded into 10% SDS–polyacrylamide gel. The segregated proteins were then transferred onto polyvinylidene fluoride membranes; blocked with 5% (w/v) skimmed milk at 25°C for 1 hour; incubated with CCR2, CXCR2, CXCR4, IL-6R, IL-1βR, and TNF-αR primary antibodies (all from Abcam) at 4°C overnight; and then further incubated with horseradish peroxidase–conjugated secondary antibody (Thermo Fisher Scientific, USA), and the blots were developed using a West Pico PLUS chemiluminescent substrate kit (Thermo Fisher Scientific, USA). The stability of the cytokine/chemokine receptors in N-NVs over 14 days and the stability of the cytokine/chemokine receptors before and after DEX loading into N-NVs were also investigated by Western blotting.

Samples were extracted and quantified following denaturation, and equivalent them were loaded into a 10% polyacrylamide gel, then transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking, the PVDF membranes were incubated with primary anti-body. Finally, the PVDF membranes were further incubated with horseradish peroxidase (HRP) conjugated secondary antibody (Thermo Fisher, CA, USA), and the blots were developed by a west Pico PLUS chemiluminescent substrate kit (Thermo Fisher, CA, USA). The following antibodies were used: anti-TNFR1 (Sino Biological, Beijing, China), Ly6G (Thermo Fisher Scientific, NY, USA), COX-2 (Proteintech, IL, USA), and β-actin antibody (Sigma-Aldrich, MO, USA).