Anti fibronectin antibody

Cells were seeded at 10 x 10⁴ cells/well into 6-well plates and incubated in presence or absence of 10 ng/ml TGF-ß1 with or without terfenadine, ebastine, and solifenacin at 10 μM for 72h. Protein was isolated using RIPA buffer (Sigma Aldrich, UK). 20 μg of protein mixed with Li-Cor 4x protein loading buffer (Li-Cor, UK) and heat denaturated at 95°C under reducing conditions. Samples were then loaded onto Any kD^• Mini-PROTEAN^® TGX Precast Protein Gels (Bio-Rad) along with a protein ladder (Bio-Rad). After gel electrophoresis, the separated protein was transferred onto a methanol-activated PVDF membrane (Millipore) by wet blotting at 350 mA for 1h. The membrane was washed and left to dry for 1h before being blocked with Li-Cor Intercept Blocking Buffer (Li-Cor) for 1h. The membranes were incubated overnight with primary antibodies (anti–α-SMA antibody, 1:3000 (Sigma-Aldrich); anti-fibronectin antibody, 1:1000 (ThermoFisher); anti-GAPDH, 1:10,000 (Abcam)) at 4°C on a shaker. Membranes were washed 4x with Tris-buffered saline containing 0.1% Tween 20 before incubation with secondary antibodies (Li-Cor, 1:10,000) for 1h on a shaker in darkness. This was followed by 4 washes with Tris-buffered saline containing 0.1% Tween 20. Blots were visualised using an infrared imaging system (Odyssey CLx imager; LI-COR) at 700 and 800 nm wavelengths simultaneously.

Piceatannol was purchased from Future Chem (Seoul, Korea). Anti-alpha smooth muscle actin (α-SMA; 1:1000, sc-130617), anti-CTGF (1:1000, sc-14939), anti-HDAC3 (1:1000, sc-11417), anti-HDAC4 (1:1000, sc-11418), anti-HDAC5 (1:1000, sc-133225), anti-TGF-β1 (1:1000, sc-146), anti-JNK (1:1000, sc-7345), anti-ERK1 (1:1000, sc-271269), and anti-GAPDH (1:1000, sc-32233) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Primary antibodies against collagen type I (1:1000, ab34710), HDAC2 (1:1000, ab12169), HDAC8 (1:1000, ab137474), and HDAC10 (1:1000, ab53096) were purchased from Abcam (Cambridge, MA, USA). Anti-fibronectin antibody (1:1000, MA5-11981) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Anti-HDAC1 antibody (1:1000, 06–720) was purchased from Merck Millipore (Darmstadt, Germany). Anti-HDAC6 (1:1000, 7612), anti-Smad3 (1:1000, 9523), anti-Smad2 (1:1000, 3103), anti-Smad4 (1:1000, 9515), anti-p-Smad3 (1:1000, 9520), anti-p-JNK (1:1000, 9251), anti-p-p38 (1:1000, 4511), anti-p-ERK1/2 (1:1000, 4370), and anti-p38 (1:1000, 8690) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).