Cells were first starved by incubation for 24 hours in serum-free medium. After serum starvation for 24 h, the cells were treated with 44 µM ATX for 24 h, with or without the ATX inhibitor, HA 130 (1 µM), which was added 30 min before ATX treatment. After 24 hrs of incubation, cells were collected in RIPA Buffer (Thermo Fisher Scientific K.K., Kanagawa, Japan) containing protease inhibitors (Roche Diagnostics, Basel, Switzerland), sonicated, and centrifuged. Protein concentrations in the supernatant were determined by a BCA assay using a BCA Protein Assay Kit (Thermo Fisher Scientific K.K., Kanagawa, Japan). Proteins were boiled in Sample Buffer (Thermo Fisher Scientific K.K., Kanagawa, Japan). The same amounts of proteins were loaded onto 4–20% precast polyacrylamide gels (BIO-RAD Laboratories, Hercules, CA USA) and separated by SDS-PAGE. Protein bands were transferred to PVDF membranes (BIO-RAD Laboratories, Hercules, CA USA) and the membranes were immersed in Tris-Buffered Saline with Tween 20 (TBST) containing the first antibody. After washing, the membranes were immersed in TBST containing the second antibody and reacted with ECL substrate (Thermo Fisher Scientific K.K., Kanagawa, Japan). Protein bands were detected by ImageQuant LAS 4000 mini (GE Healthcare, Chicago, IL USA). Primary antibodies were: anti-αSMA (Sigma-Aldrich Co., LLC St. Louis, MO USA, 1:1000) and anti-fibronectin (1:1000; Abcam, Cambridge, MA, USA), anti-collagen type I (1:1000; Cell Signaling Technology, Danvers, MA, USA). HRP-conjugated second antibody (1:10,000) was purchased from Thermo Fisher Scientific (Waltham, MA USA). β-tubulin served as the loading control. All membranes were stripped of antibodies using WB Stripping solution and incubated with mouse monoclonal antibody β-tubulin (1:1000), and subsequently with H goat anti-mouse IgG antibody (1:2000). Densitometry of scanned films was performed using ImageJ 1.49 (NIH Bethesda, MD, USA), and results are expressed relative to the loading control (β-tubulin).
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