Western Blot Protein Extraction Protocol

For protein extraction, 12.5 × 10⁴ cells per well were seeded onto 6-well plates and harvested after 72 h. Therefore, cells were washed once with cold PBS, and 56 µL of radioimmunoprecipitation assay buffer (30 mM Tris, 0.5 mM EDTA, 150 mM NaCl, 1% NP-40, 0.1% SDS) supplemented with 10% protease and phosphatase inhibitors (Hoffmann-La Roche, Basel, Switzerland) was added directly to each well and cells were scraped off. Protein concentration was determined with a bicinchoninic acid kit (Thermo Fisher Scientific, Waltham, MA USA). Western blots were performed as described previously [8 (link),20 (link)]. 10 µg of whole cell protein was separated on a 10% polyacrylamide gel and subsequently transferred to a nitrocellulose membrane. Immuno-detection was performed with a Fusion FX device (Vilber Lourmat Deutschland GmbH, Eberhardzell, Germany). Table 2 summarizes all antibodies and respective concentrations used in this study.