0.22 μm polyvinylidene difluoride membrane

Cell lysates were prepared in M-PER buffer (Thermo Fisher Scientific, 78501) supplemented with protease inhibitors (Roche). For Western blotting, cell lysates and EVs were resuspended in NuPAGE LDS sample buffer (Novex, NP0008) with NuPAGE sample reducing agent (Novex, NP0009). IP was carried out using the FLAG immunoprecipitation kit (Sigma-Aldrich). Cell lysates were incubated in anti-FLAG agarose affinity gel at 4°C overnight. Immunoprecipitated samples were washed three times with washing buffer, eluted, and subjected to Western blotting. All Western blotting samples were loaded on a 4 to 12% NuPAGE gel or 12% NuPAGE gel and transferred onto 0.22-μm polyvinylidene difluoride membrane (Bio-Rad, 1620177). Primary antibodies include anti-FLAG antibody (Sigma-Aldrich, F1804; 1:2000 dilution), anti-vinculin antibody (Abcam, ab129002; 1:2000 dilution), anti-CD9 antibody (Cell Signaling Technology, 1317S; 1:1000 dilution), anti-Scamp3 antibody (GeneTex, GTX102216; 1:2000 dilution), and anti-ARRDC1 antibody (in-house; 1:3000 dilution). Secondary antibodies include horseradish peroxidase (HRP)–conjugated anti-rabbit (Cell Signaling Technology, 7074S; 1:2000 dilution) and HRP-conjugated anti-mouse (Cell Signaling Technology, 7076S; 1:2000 dilution).

Due to the low molecular weight of chemokines IP-10 and IL-8, the protein lysates extracted from the HUVECs overexpressing lnc-SLC15A1-1 were separated by a Tris-Tricine SDS-PAGE gel electrophoresis system (cat. no. ab119197, Abcam, UK). For the Western immunoblotting assay, the target protein was transferred to a 0.22 μm polyvinylidene difluoride membrane (Bio-Rad Laboratories, Hercules, CA, USA) following the manufacturer’s instructions. The membranes were blocked in Lightning Blocking Buffer (Arrowtec, Taiwan) for 5 min and then hybridized to primary antibodies against IP-10 (cat. no. AHP782, Bio-Rad Laboratories, Hercules, CA, USA) or IL-8 (cat. no. ab52612, Abcam, UK). After immunoblotting, the membranes were washed in 0.05% Tris-buffered saline (Omics Bio, Taiwan) with Tween 20 and reacted with horseradish peroxidase-conjugated goat anti-mouse IgG (cat. no. C04001, Croyez Bioscience, Taiwan). The protein bands were visualized using an enhanced chemiluminescence system (cat. no. GTX14698, GeneTex, Irvine, CA, USA). β-actin was used as the loading control (cat. no. A5441, Sigma-Aldrich, Germany).