Rabbit anti cd9

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.

EV samples were combined with SDS sample buffer (Boston Bioproducts, Ashland, Massachusetts, USA), separated by denaturing electrophoresis on precasting 4–12% Novex or 4–12% Bolt gels (Life Technologies, Carlsbad, California, USA), and then transferred onto a nitrocellulose membrane (Life Technologies) by applying 20 V for 7 min at RT. The blots were blocked with 5% milk in TBST (tris buffered saline-tween 20; Boston Bioproducts, Ashland, Massachusetts, USA) for 1 h at RT, and incubated with rabbit anti-CD9 (1:1000; Cell Signaling Technologies, Danvers, Massachusetts, USA), rabbit anti-Rab5 (1:300; Santa Cruz Biotechnology, Dallas, Texas, USA), rabbit anti-CD63 (1:200, Santa Cruz Biotechnology, Dallas, Texas, USA), rabbit anti-aquaporin-2 (1:1000; Abcam, Cambridge, Massachusetts, USA), and rabbit anti-Hsp70 (1:1000; Abcam) antibodies in blocking buffer overnight at 4°C. After incubation, the blots were washed with TBST and incubated for 1 h at RT with goat anti-rabbit HRP antibody (1:2000; Cell Signaling Technologies) in blocking buffer. Western Bright ECL (BioExpress, Kaysville, Utah, USA) was used to develop the blots. The equivalent of 3 ml of urine EVs was loaded per lane. In Figure 3(b), the immunoblot band intensity for AQ2, CD9, and Rab5 is represented in a heat map (Excel), with red being the most intense and blue being the least intense.