Ecl plus reagent

Western blot analysis was carried out according to protocol as described previously^{30 (link), 36 (link)}. After two times the ice-cold PBS washes, cells were lysed in the ice-cold modified immunoprecipitation assay buffer (150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 20 mM Tris-HCl, PH 7.5) with addition of protease and phosphatase inhibitor mixtures. 20 μg of protein from each sample was separated on 12% polyacrylamide gels and transferred to PVDF membranes. After 5% milk/TBST blotting for 1 h, primary antibodies against specific genes of interest were applied, followed by HRP-conjugated secondary antibodies. The chemiluminescent signals were detected using ECL-plus reagent (GE Healthcare). The α-tubulin detected in the same sample was used as an equal loading control. Band density analysis was performed by Image J software. All data were normalized to the band density for tubulin.

Hippocampal neurons at 18–21 DIV were lysed after stimulation in lysis buffer that contained 20 mM Tris-Cl (pH 6.8) at 4 °C, 137 mM NaCl, 25 mM β-glycerophosphate, 2 mM NaPPi, 2 mM EDTA, 1 mM Na₃VO₄, 1% Triton X-100, 10% glycerol, 2 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, and 10 μl/ml protease inhibitor mixture (Sigma-Aldrich). Samples of culture media and lysates that contained equal amount of protein were mixed with 4× sodium dodecyl sulfate (SDS) sample buffer and denaturated by heating at 95 °C. The samples (20 μg) were subjected to 12% SDS-polyacrylamide gel electrophoresis and then electrotransferred onto polyvinylidene difluoride membranes (Immobilon-P, Millipore). Ponceau S staining was performed to locate the protein bands on Western blots. The membranes were then blocked in 10% nonfat milk in Tris-buffered saline with 0.1% Tween 20 (TBST) and incubated at 4 °C overnight with anti-β-dystroglycan (1:500; NCL-b-DG, Novocastra), anti-β-tubulin (1:1000; ab21058, Abcam), and anti-TIMP-1 (1:500; MAB580, R&D System) diluted in 10% nonfat milk in TBST. The membranes were then incubated with peroxidase-labeled secondary antibodies diluted 1:10000 in 10% nonfat milk in TBST for 1 h at room temperature. ECL Plus reagent (GE Healthcare) was used to detect horseradish peroxidase (HRP) on the immunoblots.

Cell total protein was extracted using RIPA buffer supplemented with phosphatase and protease inhibitors (Roche Diagnostics, Meylan, France). Protein concentration was determined using a BCA assay (Sigma Aldrich) and equal amounts of protein were resolved by SDS-PAGE. The blocked membrane was incubated with primary antibodies overnight at 4 °C. Bands were detected by using a horseradish peroxidase secondary antibody and visualized with ECL Plus reagent (GE Healthcare, Buc, France). β-Tubulin was used as loading control (Santa Cruz Biotechnology, Heidelberg, Germany).

Western blotting was performed as described^{31 (link)}. After samples were boiled for 10 min, the supernatants were separated in 4–20% gradient or 7% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene fluoride membranes (GE Healthcare Japan, Tokyo, Japan). The membranes were washed three times in TBST (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.05% Tween 20) for 5 min each, blocked for 30 min in Blocking-One (Nacalai Tesque, Kyoto, Japan), and washed three times with TBST for 5 min each. The membranes were then incubated for 1 h at room temperature with antibodies against α-smooth muscle actin (α-SMA) (Abcam, Tokyo, Japan; 1:500) and collagen I (Abcam; 1:500) in Can-get-signal (Toyobo, Osaka, Japan), washed with three times with TBST for 10 min each, incubated with peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG (Dako, Tokyo, Japan; 1:5,000) in Can-get-signal for 1 h, and washed three times with TBST for 10 min each. Signals were developed using ECL plus reagent (GE Healthcare Japan) and detected using an LAS imager (GE Healthcare Japan).