Methanol activated pvdf membrane

Mouse testis samples were lysed using RIPA buffer (Sigma Aldrich, St. Louis, MO, USA) containing a protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO, USA), and 10 µg/mL total protein was separated on 10% polyacrylamide gel via SDS-PAGE, followed by transfer on methanol-activated PVDF membrane (Merck Millipore, Burlington, MA, USA). The membranes were blocked in 5% non-fat powdered milk for 1 h at room temperature, after which they were incubated with primary antibodies to Akt (pS473) + total Akt Kit (ab126433, abcam, Cambridge, UK), ERK1 (phosphor) + total ERK (ab126445, abcam), p38 MAPK alpha + total p38 MAPK alpha (ab126453, abcam), cyclinD1 (ab16663, abcam) and β-actin (A5441, Sigma Aldrich, St. Louis, MO, USA), overnight, at 4 °C. The next day, after 3 washes with 0.1% PBST, the membranes were incubated with the corresponding secondary antibodies: goat α-mouse HRP or goat α-rabbit HRP (Thermo Scientific) for 1 h at room temperature. The membranes were developed with Immobilon Forte HRP Substrate (Merck Millipore, Burlington, MA, USA) using a Luminescent Image analyzer LAS-3000 (FUJIFILM, Tokyo, Japan). The relative protein expression was quantified via densitometric analysis with the Image J software (Version 1.53r).

Following SDS-PAGE^{56 (link)} of the recombinant full-length hGal3 and its fragment P113-hGal3, the polypeptides were electro-transferred onto a methanol-activated PVDF membrane (Merck Millipore, Billerica, MA, USA). After washing in phosphate-buffered saline (115 mM NaCl, 15 mM Na₂HPO₄, 4 mM KH₂PO₄; PBS) containing 0.01% v/v Tween 20 (PBS/T_0.01) the membrane was incubated with the chimeric αhGal3-Fab-PAS200-Cy5.5 (2 µg ml⁻¹ in PBS/T_0.01)^{9 (link)} for 1 h at room temperature. After washing the membrane with PBS/T_0.01, fluorescence was detected with an Odyssey fluorescence scanner (excitation: 685 nm; emission: 720 nm; LI-COR, Lincoln, NE, USA) and evaluated using Quant version 12.2 software (TotalLab, Newcastle-Upon-Tyne, UK).

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.