Anti flag tag clone m2

Melanoma cell lysates were separated on SDS-PAGE gels and transferred to PVDF membranes. After blocking with 1% BSA for 1 h, the membranes were incubated with primary antibodies at 4 °C overnight. Next day, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Blots were then developed using an enhanced chemiluminescence western blotting detection kit (BioRad, Hercules, CA, USA). Antibodies against Phospho-p44/42 MAPK (Thr202/Tyr204, clone 197G2, #4377), FOXD3 (clone D20A9, #2019), HA-tag (clone 6E2, #2367, clone C29F4, #3724), Myc-tag (clone 71D10, #2278), HER3/ErbB3 (clone 1B2E, #4754), Phospho-Akt (Ser473, clone D9E, #4060), AKT (#9272), Phospho-MAPK Substrates Motif [PXpTP] (#14378) were purchased from Cell Signaling Technology (Beverley, MA, USA). Anti-β-actin (#A2066) and anti-FLAG-tag (clone M2, #F3165) were from Sigma-Aldrich. Anti-SOX10 (N-20, #SC-17342) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Another anti-FOXD3 (#631702) antibody was purchased from Biolegend (San Diego, CA, USA).

U251-MG cells transfected with appropriate expression vectors were lysed in 1x sample loading buffer (62.5 mM Tris pH8.0, 0.2% (w/v) SDS, 0.25% (v/v) β-mercaptoethanol, 10% (v/v) glycerol; 0.01% (w/v) bromophenol blue) and the obtained lysates were separated on SDS-PAGE electrophoresis, transferred to PVDF membrane (Millipore) and analyzed by Western blot. Following antibodies were used: anti-FLAG tag (clone M2, Sigma Aldrich, St. Louis, MO, USA), anti-β-actin, anti-mouse-HRP and anti-rabbit HRP (Cell Signaling, Danvers, MA, USA). Luminescence was detected using Clarity Western ECL Substrate (Bio-Rad, Des Plaines, IL, USA) and recorded using the Fusion-Fx documentation system (Vilber Lourmat, Collégien, France).