Polyvinylidene difluoride pvdf

RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) with a protease and phosphatase inhibitor cocktail (Abcam, Cambridge, MA, USA) was used to extract total proteins from fresh mouse ears and cell samples. The nuclear proteins were separated using Pierce NE-PER Nuclear and Cytoplasm Extraction Kit (Thermo Fisher Scientific). The protein concentration was measured using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Briefly, 30 μg of protein was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), transferred to polyvinylidene difluoride (PVDF) (Millipore, Burlington, MA, USA), and then blocked with 5% skim milk (BD Bio-sciences, San Jose, CA, USA). After incubation with the primary and secondary antibodies, each blot was developed using ECL reagent (Millipore) and captured by the Amersham Image 600 system (GE Healthcare Life Sciences, Pittsburgh, PA, USA). The protein levels were normalized to GAPDH expression. Primary antibodies in the Western blot are listed in Supplementary Table S1.

VA was purchased from Sigma Chemicals Co. (St. Louis, MO, USA). VA was dissolved in dimethyl sulfoxide (DMSO). Dulbecco modified eagle medium (DMEM) medium and fetal bovine serum (FBS) were obtained from Gibco (Grand Island, NY, USA). Insulin, 3-isobutylmethylxanthine (IBMX), and dexamethasone were obtained from Sigma-Aldrich (St Louis, MO, USA). Poly-vinylidene difluoride (PVDF) was procured from Millipore (Merck KGaA, Darmstadt, Germany). The electrochemiluminescence (ECL) kit was obtained from GE Healthcare Life Sciences (Seoul, Korea).

Mice of either sex were sacrificed at P14 or P60 and spinal cords were immediately processed for protein extraction. Protein lysates (50 μg) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto a PolyVinylidene DiFluoride (PVDF) (Millipore, Billerica, MA, USA) membrane using a buffer containing 25 mM Tris base, pH 8.3, 192 mM glycine, 20% (vol/vol) methanol for 1 h at 100 V at 4 °C. Membranes were blocked for 1 h in 10% Milk/0.1% Tween/TBS, then incubated overnight at 4 °C with the primary antibody diluted 1:1000 in 5% BSA/ 0.02% sodium azide/0.1% Tween/TBS. Antibodies used are: mouse anti-CNPase (Sternberger Inc., SMI-91, 1:5,000), mouse anti-MBP (Millipore, SMI-99, 1:5,000), mouse anti-GAPDH (Abcam, ab8245, 1:5,000). After rinsing with 0.1% Tween/TBS, membranes were incubated 2 h at room temperature with the secondary light-chain specific antibody (goat anti-mouse IgG, Jackson Immunoresearch, 115-035-174, 1:10,000) in 10% Milk/0.1% Tween/TBS. After rinsing, membranes were incubated with ECL (Amersham) for 3 min and then revealed. Quantification was carried out on three biological replicates per genotype, using ImageJ. Protein expressions were normalized to GAPDH expression, then compared to their respective expression levels in control samples. Uncropped gels are shown in Supplementary Fig. 9.

Flaccidoxide-13-acetate was supplied by Dr. Jui-Hsin Su. Rabbit anti-human Akt, FAK, MMP-2, MMP-9, MMP-13, mTOR, PI3K, TIMP-1, TIMP-2, uPA and phosphorylated Akt, mTOR, and PI3K were purchased from Cell Signaling Technology (Danvers, MA, USA). E-cadherin, N-cadherin, lamin A2, and Snail antibodies were supplied by Epitomics (Burlingame, CA, USA). HRP-conjugated goat anti-rabbit immunoglobulin (IgG) was obtained from Millipore (Bellerica, MA, USA). Thiazolyl blue tetrazolium bromide, streptomycin, penicillin, and DMSO were purchased from Sigma (St. Louis, MO, USA). Goat anti-rabbit and horseradish peroxidase-conjugated immunoglobulin IgG and polyvinylidene difluoride (PVDF) membranes were obtained from Millipore (Bellerica, MA, USA). Fetal bovine serum and DMEM were purchased from Biowest (Nuaillé, France).

Monoclonal rat anti-mouse CD34 FITC-conjugated antibody, monoclonal rat anti-mouse Sca-1 PE-conjugated antibody, polyclonal rat anti-mouse CD11c PE-conjugated antibody, monoclonal rat anti mouse Qa2 antibody, monoclonal rat anti mouse IDO antibody, polyclonal rat anti mouse β-Actin HRP-conjugated antibody, monoclonal chicken anti rat HRP-conjugated antibody (all from Abcam, Cambridge, MA, USA), Dulbecco’s modification of Eagle medium (DMEM), Roswell Park Memorial Institute (RPMI), fetal bovine serum (FBS), enhanced luminol-based chemiluminescent substrate (ECL), Granulocyte-macrophage colony stimulating factor (GM-CSF), Trizol, RIPPA buffer, Bradford kit, protein inhibitor, polyvinylidene difluoride (PVDF) (all from Sigma, Ronkonkoma, NY, USA), cDNA synthesis kit (TAKARA, Japan), PCR SYBR Green kit (TAKARA, Japan), Nycodenz (Axis shield, Norway), Pan DC Microbead MACS (Miltenyibiotec, Germany).

Pyrrole monomer (C₄H₅N) (99%, Sigma-Aldrich, Bengaluru, India), Ferric chloride (FeCl₃) (97%, Sigma-Aldrich, Bengaluru, India), Tin (IV) chloride pentahydrate (SnCl₄·5H₂O) (98%, Sigma-Aldrich, Bengaluru, India), Hydrazine hydrate (N₂H₄·H₂O) (98%, Merck Chemicals, Bengaluru, India), graphite powder (99.99%, Sigma-Aldrich, Bengaluru, India), and Polyvinylidene difluoride (PVDF) (99%, Sigma-Aldrich, Bengaluru, India) were used without further purification. Potassium Hydroxide (KOH), N-Methyl-2-pyrrolidone (NMP), Hydrogen peroxide (H₂O₂), and activated carbon were purchased from Merck Chemicals, Bengaluru, India. Nitric acid (HNO₃) and Sulphuric acid (H₂SO₄) were purchased from Merck Chemicals, Bengaluru, India (99.99%), in addition to Polyvinyl Alcohol (PVA) being obtained (99%, Sigma-Aldrich, Bengaluru, India). All the reagents were used as received without any purification. Ultra-pure water was used throughout the experiments, which was attained from a Milli-Q system from Millipore (Milford, MA, USA).