Col1a1

Total cell lysates were generated using a lysate buffer (150 mM NaCl, 0.1% SDS, 1% Triton x-100, 0.5% Sodium Deoxycholic and 50 mM Tris, pH 8.0). Appropriate amount of protease and phosphatase inhibitors were added just before use. 4–12% Tris gel were poured and equivalent amount of lysate samples were loaded and electrophoresed. The run gels were transferred to nitrocellulose membrane and blocked for 1 h at room temperature in Tris-buffered saline containing 0.1% Tween-20 (TBS-T)/5% nonfat dry milk (Bio-Rad Laboratories). The membranes were then incubated with specific primary antibodies (ZEB1 (Santa Cruz Biotechnology, INC), COL1A1 (Santa Cruz Biotechnology, INC), α-SMA (Sigma Aldrich) and GAPDH (Abcam) overnight at 4⁰C. The blotted membranes were washed several times with TBS-T, then incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at RT. Positive proteins were visualized by enhanced chemiluminescence (ECL) and exposure to film (Kodak, UK).

Western blots were conducted as previously described [18 (link)]. Total protein was extracted using the radioimmunoprecipitation assay buffer. The extract was centrifuged (14,000 rpm) at 4 °C for 30 min. The supernatant was collected, and protein levels were quantified using the Bradford assay. Proteins were separated via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane for 2 h. Subsequently, the membrane was blocked with 3% BSA in phosphate-buffered saline containing 0.1% Tween 20 (PBST) for 2 h at room temperature and incubated with primary antibodies at 4 °C overnight. After washing thrice with PBST, the membrane was incubated with secondary antibodies at 37 °C for 4 h. Antibodies against β-catenin, p-Smad1/5, and p-Smad2/3 and secondary anti-rabbit antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA), and BMP2/4, TGFβ, RUNX2, OSX, OPN, Col1a1, β-actin and secondary anti-mouse antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Bands were detected using a Pierce^® ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA, USA). The resulting bands were normalized using β-actin as a loading control.

After cultivation with different EPTs for 8 and 16 days, the total protein was extracted from the cells using M-PER protein extraction reagent (Pierce). The protein concentration was measured by BCA Protein Assay Kit (Pierce) according to the manufacturer’s instructions. Proteins were run on SDS-PAGE gel and transferred in to a PVDF membrane (Millipore, MA, USA) according to the standard protocol. After blocking in 5% skim milk for 2 hours at room temperature, the membrane was incubated with primary antibodies against Col1a1, OPN, BSP, OC and OSX (Santa Cruz Biotech) overnight at 4 °C, and then incubated in the solution of secondary antibodies for 1 hour at room temperature. β-actin (Santa Cruz Biotech) was used as the internal control. Protein bands were captured using ECL detection kit (Pierce).

Total protein was isolated from tissues using mammalian protein extract buffer (GE Life Science, 28-9712-79) containing a protease inhibitor cocktail (Sigma-Aldrich, P8340). Equal quantities of protein were separated and transferred onto polyvinylidene fluoride membranes. The membranes were then incubated with one of the following antibodies, as appropriate, at a concentration of 1 in 1,000: anti-β-actin (Cell Signaling, #8457), fibronectin (Santa Cruz, sc-8422), COL1A1 (Santa Cruz, sc-293182), CTGF (Santa Cruz, sc-101586), TGF-β (Cell Signaling, #3711), and anti-lipocalin-2/NGAL antibody (Abcam, ab63929). The density of each band was quantified using ImageJ and normalized to β-actin.