Epr2949

After isolation, we lysed the exosomes in lithium dodecyl sulphate buffer (LDS buffer) and measured the protein concentration by BCA protein assay kit. Later, protein extracts were separated on SDS-PAGE, transferred to a PVDF membrane, and blocked with 5% milk in PBS and incubated overnight at 4°C with primary antibodies for CD9 (Abcam; EPR2949, 1:2000), CD63 (Santa Cruz Biotechnology; MX-49.129.5, 1:200), and HSP70 (Santa Cruz Biotechnology; sc-137210, 1:200). After washing, the membranes were incubated with the horseradish peroxidase-conjugated secondary antibody (Nanjing Jiancheng, 1:1000) for 1 h and washed again. Lastly, membranes were developed and images were collected on Bio-Rad Molecular Imager.

Proteins were extracted using RIPA Lysis Buffer (Thermo Fisher Scientific, MA, USA) containing protease inhibitors cocktail (Cell Signaling Technology, MA, USA). Protein concentration was measured using the BCA Protein Assay Kit (Beyotime Institute of Biotechnology). Then, protein extract (20 μg) was separated by SDS-PAGE and transferred onto polyvinylidene fluoride microporous membranes (Merck Millipore, Darmstadt, Germany). The membrane was blocked with 5% milk in 0.5% Tris-buffer saline solution (pH 7.6) for 1 h, then incubated overnight at 4 °C with primary antibodies for CXCR4 (abcam; UMB2, 1:1000), GAPDH (abcam; EPR16891, 1:5000), CD9 (abcam; EPR2949, 1:2000), CD63 (Santa Cruz; MX-49.129.5, 1:200), TSG101 (abcam; EPR7130(B), 1:1000), or actin (abcam; EPR16769, 1:5000). The membrane was then incubated at room temperature for 30 min with HRP-conjugated secondary antibodies. The image data were collected on Bio-Rad molecular Imager with Image Lab^TM Software and analyzed with NIH ImageJ.