Rabbit anti syntenin

For analysis of protein levels in EVs, isolated EVs were lysed with RIPA buffer, followed by addition of 1X Laemlli buffer and incubation at 95°C for 5 min. Samples were subject to standard Western blotting with chemiluminescence detection. The following antibodies were used as recommended by the manufacturer: rabbit anti‐Src (Cat#: 2109, 1:1000), rabbit anti‐calnexin (Cat#: 2679, 1:5000), rabbit anti‐CD‐9 (Cat#: 13403 for human species, 1:1000), rabbit anti‐GAPDH (Cat#: 2118, 1:10,000), rabbit anti‐gamma‐tubulin (Cat#: 5886, 1:1000), and mouse anti‐Cas9 (Cat#: 14697, 1:1000) were all purchased from Cell Signaling Technology. Mouse anti‐VSV‐G (Cat#: EB0010, Kerafast, 1:1000), rabbit anti‐syntenin (Cat#: ab19903, Abcam, 1:1000), mouse anti‐CD63 (Cat#: 556019, BD Pharmingen, 1:500). Secondary antibodies anti‐rabbit IgG HRP (Cat#: 7074, 1:5000), anti‐mouse IgG HRP (Cat# 7076, Cell Signaling Technology, 1:5000). The anti‐myristoylated octapeptide antibody described in this study was used at dilutions of 1:250, 1:500, or 1:1000. Cas9 recombinant protein was purchased from Sigma Aldrich (Cat# Cas9Prot). EV protein analysis was done in accordance with the MISEV 2018 guidelines (Théry et al., 2018 (link)). The band intensity was quantified by Image J software.

Goat anti-Alix (1:1000, St.Cruz), rabbit anti-CD147 (1:1000, #sc-13976, St.Cruz), mouse anti-GAPDH (1:2000, CB1001, Millipore) mouse anti-hsc70 (1:1000, St.Cruz), rabbit anti-SMPD2 (1:1000, HPA018125, Sigma Prestige), rabbit anti-SMPD3 (1:1000, #sc-67305, St.Cruz), rabbit anti-Tsg101 (1:1000, Sigma Prestige or 1:1000, #sc-7964, St. Cruz for Western blots), rabbit anti-syntenin (1:2000, Abcam #133267), mouse anti-Tubulin (1:5000, #05–829, Millipore), rat anti-Wnt5A (1:2000, #MAB645, R&D), mouse anti-CD81 and CD81-FITC (WB: 1:1000, FACS: 1:500 Biolegends), mouse anti-CD9-APC, mouse anti-CD147-APC, Streptavidin-FITC, mouse IgG-FITC, IgG-APC (all 1: 500, Immunotools), secondary rat, rabbit and mouse HRP (1:5000, #sc-2004, #sc-2005, #sc-2006, St.Cruz).