Nitrocellulose transfer membrane

STS cells (SW982 and Fuji) were seeded in T25 flasks (Sarstedt) and treated with panobinostat or vorinostat for 48 h. After detaching using trypsin-EDTA, total cellular proteins were extracted using RIPA buffer (Thermo Fisher Scientific) supplemented with a complete protease inhibitor cocktail (Sigma-Aldrich) for 30 min on ice. Quantification of the extracted proteins was performed by the BCA method using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Protein was diluted with 4 × loading buffer (Thermo Fisher Scientific) and heated up to 95 °C for 5 min. A total of 10 μg of protein was separated by NuPAGE 4–12% Bis-Tris protein Gel (Thermo Fisher Scientific) and transferred to a nitrocellulose transfer membrane (Thermo Fisher Scientific). Incubation with primary antibodies was performed overnight at 4 °C. Immunoblots were washed with PBST at room temperature and incubated with secondary antibodies for 1 h. Signals were detected using the ECL Prime Western blotting detection reagent (GE Healthcare, Little Chalfont, UK) and visualized by an Amersham Imager 600 (GE Healthcare). Data were analyzed using ImageJ (NIH, Bethesda, MY, USA).

The sEVs were lysed in RIPA buffer with a protease inhibitor cocktail (Complete ULTRA, Roche, Switzerland), a phosphatase (PhosSTOP, Roche, Switzerland), and sonicated. The proteins (30 μg per well) were loaded onto 8% or 10% SDS polyacrylamide gels and subjected to electrophoresis. Separated proteins were transferred to a 0.45 µm Nitrocellulose Transfer Membrane (Thermo Fisher Scientific) using the Mini Trans-Blot Cell Module (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Membranes were incubated overnight at 4 °C with the appropriate primary antibodies (CD9 #10626D, CD63 #10628D, and CD81 #10630D purchased from Invitrogen, Integrin α5/CD49e #610633 from BD Biosciences, and Integrin β1 Antibody #4706S from CST). The applied dilutions are shown in Supplementary Materials Table S1. A primary monoclonal antibody against β-actin (# A1978 Sigma-Aldrich, St-Louis, MO, USA) served as a loading control. The specific binding of the antibodies was detected with the appropriate peroxidase-conjugated Alexa Fluor 680 secondary antibody and visualized using an LI-COR^® instrument (LI-COR^®, Lincoln, NE, USA).