Vin 11 5

Total protein was extracted from purified SCs or sciatic nerves that were washed with PBS 3 times and dissociated in lysis buffer containing 10 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA-2Na, 1% Nonidet P-40, 1 mM Na₂VO₄, 10 mM NaF and Protease Inhibitor Cocktail (11873580001, Roche) for 30 min on ice. The supernatants were collected by centrifugation. Total protein was denatured and used for western blotting. The following primary antibodies were used in western blots: anti-rabbit Myelin Protein Zero (ab31851, Abcam), anti-rat myelin basic protein (MAB386, Merck Millipore), anti-mouse MAG (sc-166848, Santa Cruz Biotechnology), anti-rabbit TRPV4 (CB-ACC-034, Alomone), anti-rabbit EGR2 (ab108399, Abcam), anti-rabbit c-Jun mAb (9165, Cell Signaling Technology), and anti-mouse vinculin antibody (VIN-11-5, Sigma). The following secondary antibodies were used for western blotting: HRP-conjugated GAPDH rabbit mAb (8884, Cell Signaling Technology), HRP-linked anti-rabbit IgG (7074, Cell Signaling Technology), HRP-linked anti-mouse IgG (7076, Cell Signaling Technology), and HRP-linked anti-rat IgG (112-035-062, Jackson).

Cell lysates were obtained in radioimmunoprecipitation assay (RIPA) buffer containing 1 mM of PIC (Protease Inhibitor Cocktail, SIGMA), 10 mM of NaF, 1 mM of Na₃VO₄, and 0.5 mM of DTT. Cell lysate measuring 10 μg was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto an Immobilon polyvinylidene difluoride membrane (Millipore). Protein detection was carried out using mouse monoclonal Ab against IκBα (clone L35A5, #4814; Cell Signaling), rabbit polyclonal against p-p38 (clone D3F9), pJNK (clone 81E11) and pERK (clone D13.14.4E, #9910; Cell Signaling), p-IRF3 (clone 4D4G, #4947; Cell Signaling), and p-STAT1 (clone D4A7, #7649; Cell Signaling). Protein loading was normalized using an antibody against GAPDH (clone 6C5, sc-32233, Santa Cruz Biotechnology) or against human Vinculin (clone VIN-11-5, #V4505; Sigma-Aldrich).