Trans blot turbo mini 0.2 μm nitrocellulose transfer packs

Proteins were extracted in RIPA lysis buffer (0.05 M Tris-HCl, pH 7.4, 0.15M NaCl, 0.25% deoxycholic acid, 1% NP-40, 1 mM EDTA) freshly supplemented with 1X protease inhibitors and 1X phosphatase inhibitors. The cells were extracted with 10 sec of sonication and then on ice for 30 min. The lysates were centrifuged and the supernatants were transferred into new vials. Protein concentration was measured using the BCA assay Pierce method.
Samples were boiled (90°C) for 5 min in classical Laemmli buffer prior to western blotting. Twenty micrograms per lane of total proteins were separated by a gel 10% Mini-PROTEAN® TGX Stain-Free™ (tris glycine) Protein Gels (Biorad, Marne La Coquette, France) at 150 V for about 50min using the Mini-Protean III™ device (Bio-Rad) and then transferred to a Trans-Blot® Turbo Mini 0.2 μm Nitrocellulose Transfer Packs (Bio-Rad) using the Transblot-Blot® SD Semi-Dry Transfer Cell (Bio-Rad) at constant 25V for 7 min.
Membranes were then incubated with primary antibodies overnight (4°C) (S2 Table). Bound antibodies were detected by using the WesternBreeze Immunodetection Chemiluminescent System (Invitrogen, Fisher Scientific, Illkirch, France). The optical density of bands was visualized with the Image System (ChemiDoc, Biorad) and analyzed with Quantity one-image analysis software (Bio-Rad Laboratories).

EV pellet expression of proteins including cytokines, placental marker (hPL), TMPRSS2, ACE2, FOXP3, CD63, and CD81, was tested by western blot (Supplementary Table S1). Briefly, 30 ul of EV pellets obtained from similar PPP volumes (250ul) were combined with a 2xlysis buffer (RayBiotech) supplemented with 1% proteinase inhibitor and 1% phosphatase inhibitors (Sigma) containing β-mercaptoethanol (1:20, Biorad). Samples were loaded and separated on 4%–20% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad) and then transferred to Trans-Blot Turbo Mini 0.2 μm Nitrocellulose Transfer Packs (Bio-Rad). The membranes were stained with Ponceau S solution (P7170, sigma) to ensure protein transfer from the gel to the membrane. Membranes were washed and immunoblotted. The membranes were incubated with specific antibodies, documented by myECL™ Imager and analyzed by My Image Analysis Software (both from Thermo Fisher Scientific, Waltham, MA United States).