Anti β actin

Isolated sEVs (27 × 10⁷) were centrifuged and resuspended in 40 µL of RIPA buffer followed by a 20-minute freeze-thaw cycle. sEVs were harvested and centrifuged at 14,000 rpm (15 minutes at 4^◦C). The supernatant was transferred to a new microtube for precipitation. Cell protein (50 µg/200 µl lysis buffer) from TEX or EX were incubated with 15 µg biotinylated anti-Gal3BP for 1 hour at 4°C with constant shaking. The complex was precipitated using streptavidin magnetic beads (µMACS Streptavidin Kit; Miltenyi Biotec) and placed in a microcolumn under the magnetic field of a μMACS separator. The column was rinsed and the target molecules that bonded to the biotinylated probe were eluted. The eluted samples were subjected to SDS-PAGE on a 10% gel. Western blotting was performed using anti-mouse Gal3BP (2 µg/ml; Novus Biological) or biotinylated Gal3 (0.1 µg/ml; R&D Systems), followed by incubation with goat anti-mouse 680RD (Li-Cor) or streptavidin 800CW (Li-Cor) for 2 hours at room temperature. After washing, proteins were visualized using a Li-Cor Odyssey infrared imager. The data were normalized to anti-β-actin (R&D Systems) to determine the percentage of protein expression.

Cells were treated as aforementioned, MCF-7 cells (1.0×10⁶) were lysed in radioimmunoprecipitation assay buffer containing 1 mM phenylmethanesulfonyl fluoride (Beyotime Institute of Biotechnology, Nanjing, China). The protein concentrations were determined using a NanoDrop 2000 Micro-volume spectrophotometer (Thermo Fisher Scientific, Inc., Wilmington, DE, USA). A total of 200 µg protein in each lane was electrophoresed using 12% SDS-PAGE, and the gels were transferred onto a polyvinylidene fluoride membrane. Subsequently, membranes were blocked with 5% skim milk at room temperature for 1 h, and incubated with primary antibodies at 4°C overnight. Then the antibody-bound membranes were washed three times, each time for 10 min. The HRP conjugated goat anti-rabbit secondary antibody (cat. no. A0208; dilution, 1:1,000; Beyotime Institute of Biotechnology) incubated with membranes at 37°C for 1 h. After washed three times, the membranes were visualized by using LuminataTM Crescendo Western HRP substrate (EMD Millipore, USA). Primary antibodies were as follows: Anti-STAT3 (cat. no. MAB1799; dilution, 1:500), anti-phosphorylated (p)-STAT3 (cat. no. MAB4607; dilution, 1:500; R&D Systems, Minneapolis, MN, USA), anti-β-actin (cat. no. sc47778; dilution, 1:1,000) and anti-B-cell lymphoma-associated X (Bax) (cat. no. sc493; dilution, 1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA).