Mouse anti erk1 2

Cells were lysed in RIPA buffer (100 mM Tris–HCl, pH 7.5, 300 mM NaCl, and 0.2% SDS) containing 1% IGEPAL CA-630 and the Halt™ Protease Inhibitor Cocktail (Thermo Fisher Scientific) with subsequent centrifugation (18,000 × g, 4 °C, 20 min). The total protein concentration in the supernatants was quantified using the BCA method (Pierce BCA Protein Assay; Thermo Fisher Scientific). Samples with equal total protein content were loaded and separated on SDS–PAGE gels (Mini-PROTEAN TGX Stain-Free Gels; Bio-Rad Laboratories, Hercules, CA, USA) and then transferred onto nitrocellulose membranes (Bio-Rad Laboratories). We used the following primary antibodies: rabbit anti-NMU, rabbit anti-NMUR1 (Genetex, Irvine, CA, USA), mouse anti-IGFBP-7 and mouse anti-vimentin (Santa Cruz Biotechnology, Dallas, TX, USA). For the analysis of ERK1/2 levels, mouse anti-ERK1/2 (Santa Cruz Biotechnology) and rabbit anti-pERK1/2 antibodies (Thermo Fisher Scientific) were used. GAPDH or α-tubulin was used as a loading control and detected using rabbit anti-GAPDH or mouse anti-α-tubulin antibodies, respectively (Abcam, Cambridge, GB). HRP-conjugated goat, anti-mouse (Santa Cruz Biotechnology) or anti-rabbit secondary antibodies (Thermo Fisher Scientific) were used. The signal was detected by measuring the chemiluminescence (Thermo Fisher Scientific) with Kodak BioMax Light Film from Eastman Kodak (NY, USA).

For immunoblotting experiments, the following primary antibodies were used: rabbit anti-HA 1∶1000 (Sigma), mouse anti-TfR (transferrin receptor) 1∶400 (Invitrogen), rabbit anti-Cav-1 (caveolin-1) 1∶250 (Santa Cruz Biotechnology), mouse anti-α-tubulin 1∶16000 (Sigma), mouse anti-ERK1/2 1∶1000 (Santa Cruz Biotechnology) and mouse anti-phosphoERK1/2 (Tyr204) 1∶1000 (Santa Cruz Biotechnology), rabbit anti-Akt1/2/3 1∶4000 (Cell Signaling) and rabbit anti-phosphoAkt (Thr308) 1∶4000 (Cell Signaling). HRP (horseradish peroxidase)-conjugated donkey anti-rabbit and sheep anti-mouse secondary antibodies 1∶5000 (GE Healthcare) were used.

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).