Sc3223

Tissue and cell lysates were prepared by using RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA) with 1% Halt Protease and Phosphatase Inhibitor Cocktail (Roche, Indianapolis, IN, USA). Protein samples were separated on NuPAGE 4–12% Bis-tris gels (Invitrogen, Carlsbad, CA, USA) and transferred on PVDF membranes. Membranes were blocked with 5% non-fat milk in PBS containing 0.05% Tween 20 and probed with antibodies against ABCD1 (Abcam, ab197013, 1:5000), GFAP (Abcam, ab7260, 1:1000), CGRP (Santa Cruz, sc57053, 1:500) and peripherin (Merke, MAB1527, 1:500). Anti-β-ACTIN (Santa Cruz, SC-47,778, 1:1000) and anti-GADPH (Santa Cruz, sc3223, 1:1000) were used as a protein loading control. Membrane protein signals were prepared by using SuperSignal West Pico Chemiluminescent Substrate (Thermo, Rockford, IL, USA) after incubation with HRP-conjugated secondary antibodies.

Cells were lysed on ice in RIPA buffer containing 1 mM dithiothreitol (DTT) (Sigma-Aldrich, St Louis, MO, USA), 1× protease inhibitor (Roche, Basel, Switzerland), 1× phosphatase inhibitor (Sigma-Aldrich, USA), and 1 mM phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, St. Louis, MO, USA) before being centrifuged at 13,000 rpm for 10 min at 4 °C. The supernatants were subjected to Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA) for total protein analysis before being separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto an Immobilon-PSQ 0.2 μm Polyvinylidene Fluoride (PVDF) Membrane (Merck Millipore, Burlington, MA, USA). Blots were probed with VEGFR2 antibody (rabbit polyclonal, ab1661, Abcam, UK, RRID:AB_883437), p-ERK antibody (rabbit monoclonal, 4376, CST, USA, RRID: AB_331772), t-ERK antibody (rabbit polyclonal, 9122, CST, USA, RRID: AB_ 823567), p-AKT antibody (rabbit monoclonal, 9271, CST, USA, RRID: AB_329825), t-AKT antibody(rabbit polyclonal, 9272, CST, USA, RRID:AB_329827) and GAPDH antibody (mouse monoclonal, sc-3223, Santa Cruz Biotechnology, Inc., Dallas TX, USA, RRID:AB_627679), followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (Bethyl Laboratories, Inc., Montgomery, TX, USA).