Anti flotilin 1

Western blotting and immunofluorescence analyses were performed as published23 (link) with the following antibodies: anti-OCT4 (Abcam, Cambridge, UK), anti-Tra-1-60 (Merck Millipore, Darmstadt, Germany), anti-NANOG (Stemgent, Lexington, USA), anti-SSEA-4 (Merck Millipore, Darmstadt, Germany), anti-TUJ1 (Covance Inc., Princeton, USA), anti-Tbr1 (Abcam, Cambridge, UK), anti-Brn2 (Santa Cruz), anti-ε-sarcoglycan (esg2-1355, published antibody against the brain-specific isoform of the protein)24 (link), anti-FLAG (Sigma Aldrich, St. Louis, USA), anti-Flotilin-1 (Cell Signaling Technology, Danvers, USA), and anti-β-actin (Sigma Aldrich, St. Louis, USA).

CRC cells were lysed with RIPA buffer (100 mM TRIS-HCl, pH 7.5, 300 mM NaCl, 0.2 % SDS with 1 % IGEPAL CA-630 and the Halt™ Protease Inhibitor Cocktail (Thermo Fisher Scientific)) with subsequent centrifugation (18,000 x g, 4 °C, 20 min). Total protein concentration in lysate was quantified by BCA method (Pierce BCA Protein Assay; Thermo Fisher Scientific). Samples were loaded and separated by SDS-PAGE (Mini-PROTEAN TGX Stain-Free Gels; Bio-Rad Laboratories, CA, USA) and transferred onto nitrocellulose membranes (Bio-Rad Laboratories). NMU protein was detected with rabbit anti-NMU antibody (Genetex, CA, USA), for ERK1/2 kinases analysis mouse anti-ERK1/2 (Santa Cruz Biotechnology, USA) and rabbit anti-pERK1/2 antibodies (Invitrogen) were used. In EVs (extracellular vesicles) purity analysis we used rabbit antibodies anti-GM130, anti-annexin V and anti-flotilin-1 (Cell Signaling Technologies, Danvers, MA, USA). Proteins used as loading controls were detected by mouse anti-β-actin, mouse anti-α-tubulin or rabbit anti-GAPDH antibody (Abcam, Cambridge, GB). We used secondary HRP-conjugated goat anti-mouse (Santa Cruz Biotechnology) or anti-rabbit antibodies (Invitrogen). The signal was detected by chemiluminescence (Thermo Fisher Scientific) with Kodak BioMax Light Film from Eastman Kodak (NY, USA).