Hpa003456

Cells were washed twice with PBS containing CaCl₂ and then lysed in a 100 mM NaCl, 1% NP40, 0.1% SDS, 50 mM Tris pH 8.0 RIPA buffer supplemented with a complete protease inhibitor cocktail (Roche, Mannheim, Germany) and phosphatase inhibitors (Sigma‐Aldrich). Protein expression was examined by Western blot using the anti‐ZEB1 (1/200, Sigma HPA027524, RRID:AB_1844977), anti‐ZEB2 (1/500, Sigma HPA003456, RRID:AB_10603840), anti‐MITF (clone C5, ab80651, 1/500, Abcam, RRID:AB_1603129), anti‐SOX10 (Santa Cruz, sc‐365692, RRID:AB_10844002) antibodies for primary detection. Loading was controlled using the anti‐GAPDH (1/20,000, Millipore) antibody. Horseradish peroxidase‐conjugated rabbit anti‐mouse, goat anti‐rabbit, and donkey anti‐goat polyclonal antibodies (Dako, Glostrup, Denmark) were used as secondary antibodies. Western blot detections were conducted using the Luminol reagent (Santa Cruz).

Protein extraction and Western blotting were performed exactly as previously described [32 (link)]. The used antibodies were as follows: mouse monoclonal TS29 clone (1/500) for human Tspan8 detection; sc-166476 mouse antibody (1/500, Santa Cruz, CA, USA) for human SNAI2 detection; rabbit monoclonal antibody HPA027524 (RRID:AB_1844977, Sigma, Burbank, CA, USA) for human ZEB2 detection; rabbit monoclonal antibody HPA003456 (1/500, RRID: AB_10603840, Sigma, USA) for human ZEB1 detection; custom antibody elaborated from rabbit serum before and after immunization (Covalab, Bron, France) for Medaka Tspan8 detection; mouse monoclonal anti-actin clone C4 antibody (1/5000; MAB1501, Millipore, Darmstadt, Germany) for both human and medaka β-actin detection. The HRP-conjugated secondary antibodies were a mouse anti-rabbit IgG (1/2500, sc-2492, Santa Cruz, CA, USA) and a mouse IgG (1/2500, sc-2025, Santa Cruz, CA, USA). Western blot detections were performed using the Clarity Western ECL Substrate (Bio-Rad, Hercules, CA, USA). Digital Imaging was performed with the ChemiDoc MP Imager (Bio-Rad, Hercules, CA, USA). Western blot quantifications were achieved using the ImageJ software (1.8.0). At least two independent biological replicates were performed.