To establish the purity of the BDEVs, we examined the expression of positive markers: Hsp70 (SAB4200714, Sigma-Aldrich, St. Louis, MO, USA), Flot-1 (abcam41927, Abcam, Cambridge, UK), CD81 (MCA1846, Bio-Rad Laboratories, Inc., Hercules, CA, USA), CD63 (BD551458, BD Biosciences, Franklin Lakes, NJ, USA), HSP90 (C45G5, Cell Signaling Technology, DANVERS, MA, USA), and negative marker GM130 (BD610822, BD Biosciences, Franklin Lakes, NJ, USA), as described in previous papers [11 (link),14 (link),15 (link)]. Briefly, 25–30 μg of BDEV was subjected to Western blot under reducing (Hsp70, Flot-1, GM130) and non-reducing (CD81, CD63, HSP90) conditions using 4–12% Bis-tris gels (Invitrogen, Waltham, MA, USA).
For YWHAH validation, 15 μg of BDEV was subjected to Western blot under reducing conditions using 10% Bis-tris gel (Invitrogen, Waltham, MA, USA) against YWHAH (15222-1-AP, Proteintech, Rosemont, IL, USA). Primary and secondary antibody dilutions were carried out according to the manufacturer’s suggestions. Blots were developed using Azure CSeries Imager (Azure Biosystems, Dublin, CA, USA) with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA).
For YWHAH validation, 15 μg of BDEV was subjected to Western blot under reducing conditions using 10% Bis-tris gel (Invitrogen, Waltham, MA, USA) against YWHAH (15222-1-AP, Proteintech, Rosemont, IL, USA). Primary and secondary antibody dilutions were carried out according to the manufacturer’s suggestions. Blots were developed using Azure CSeries Imager (Azure Biosystems, Dublin, CA, USA) with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA).
Free full text: Click here
