Figure 4

Western blot and lectin detection of glycoproteins from SKOV3 cellular extracts (Ext) and secreted vesicles (Ves). (A) Con A, SNA and MAL lectin detection in SKOV3 cellular extract and vesicles. Three μg total protein were applied per lane. As positive controls, 200 ng ribonuclease B (RNase B) [41], human plasma transferrin (TFR) [42] and erythropoietin (EPO_BRP) [43] were used. (B) SNA and MAL lectin analysis of desialylated SKOV3 cellular extracts and vesicles. Total proteins (3 μg) were digested with neuraminidases from V. cholerae (Vb), A. urefaciens (Au) and S. pneumonia (Sp). Input consisted of cellular extracts and exosomes without treatment. As loading control L1 was detected. (C) Vesicles from 1.5 × 10⁷ SKOV3 cells were fractionated in a sucrose gradient. Cellular extracts (Ext), secreted vesicles from 100,000 × g pellet (Ves), pooled fractions 2-5 (F2-5) (3 μg total protein), 20% of F1, F6-7, F8-11 and F12 were analysed. As positive control for exosomes, CD9 was detected. Detection was performed using the chemiluminescent method.