Src antibody

Thawed plasma samples were centrifuged at 1,600 × g for 15 min at RT. EVs were isolated with acoustic seed trapping as described above or by centrifugation at 20,000 × g for 1 h and released or resuspended in PBS. SRC antibody (#2108, Cell signaling) or IgG isotype control antibody were added at a 1:200 dilution and the samples were incubated at RT for 1 h. EVs were then pelleted by centrifugation at 20,000 × g for 1 h and resuspended in PBS with 0.5% BSA and an Alexa Fluor 488-conjugated secondary antibody (Cell Signaling) diluted 1:1,000. The sample was incubated 30 min at RT and washed with PBS containing 0.5% BSA. The vesicle pellet was resuspended in PBS and analyzed on an Accuri C6 flow cytometer. As previously described, the EV gate was set based on a series of submicron size standard beads as described previously¹⁹ , and the proportion of SRC⁺ events in the EV gate was assessed.
For analysis of the cellular origin of SRC⁺ vesicles, plasma samples from healthy controls were prepared as described above and in addition to SRC, were stained using PE-conjugated antibodies towards CD42a, CD62E, CD16 or an isotype control (all from Beckton Dickinson, Franklin Lakes, USA) at the concentrations recommended by the manufacturer.

K562 and KA were treated with different concentrations of DMP or at different time points. After adding the lysate (50 mM NaCl, 5 mM EDTA, 0.5% SDS, 0.1 mM sodium orthovanadate, 50 μg/mL aprotinin, 1 mM phenylmethysulfonyl fluoride, and 10 mM Tris-HCl; pH 7.4), shock lysis was performed on the ice bath for 30 min, followed by ultrasonic nucleation on the ice bath for 30 seconds (50% strength, 2 s/4 s), centrifugation at 12000 rpm at 4°C for 15 min. After the supernatant was taken, the protein was quantified and SDS-PAGE gel electrophoresis was performed. After electrophoresis, the samples were transferred to nitrocellulocellulose membrane, followed by an immune reaction, and then incubated with c-Abl antibody (Cell Signaling Technology #2862), SRC antibody (Cell Signaling Technology #2108), β-actin (Cell Signaling Technology #8457) at room temperature for 4°C overnight. Goat anti-rabbit IgG-HRP (Cell Signaling Technology) was incubated for 2 hours, TBST was washed for 2 hours, and ECL solution was added for 1 minute. The membrane was drained and exposed in a bio-RAD chemiluminescence imager for several minutes. The results were read by ImageLab 5.2.1 software and analyzed statistically with β-actin as the internal reference.

Cells were washed twice with ice-cold PBS and then lysed in RIPA buffer (Beyotime Biotechnology) mixed with PMSF (100×), protease inhibitor cocktail (100×, Cell Signaling Technology), and PhosSTOPEASYpack (Roche). The reaction mixture was centrifuged at 12,000 × g for 15 min to remove cell fragments. Primary antibodies used were GBP5 Polyclonal Antibody (Proteintech, 13220-1), MMP3 Antibody (Proteintech, 17873-1), Phospho-Src Antibody (Cell Signaling Technology, 2105S), Src Antibody (Cell Signaling Technology, 2108S), Phospho-p44/42 MAPK (Erk1/2) Antibody (Cell Signaling Technology, 4370S), p44/42 MAPK (Erk1/2) Antibody (Cell Signaling Technology, 4695S), and GAPDH Antibody (Proteintech, 60004-1).