Protein ladder

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.

All chemicals were obtained from Sigma Aldrich (St. Louis, MO), unless otherwise stated. Restriction enzymes and DNA polymerases were purchased from New England Biolabs (Ipswich, MA) and were used according to manufacturers’ procedures. Protein ladder was purchased from Biorad (Hercules, CA).

Aspartame was obtained from Sigma Aldrich, St. Louis, MO, USA. All major chemicals were obtained from Himedia Laboratories Pvt. Ltd., Mumbai, India. All other chemicals were of analytical grade and obtained from Sisco Research Laboratory, Bombay, India. Anti-Goat IgG HRP conjugated (HAF017) and Goat Anti-Rabbit IgG (111-035-003) antibodies were purchased from Jackson ImmunoResearch Laboratories, PA, USA. Super Signal West Femto (34095) was purchased from Thermo Fisher Scientific, Waltham, MA, USA. Alexa Fluor 488 Goat Anti-Mouse IgG (A11001), Alexa Fluor 594 Goat Anti-Rabbit IgG (A11012), and Alexa Fluor 488 Goat Anti-Rabbit IgG (A11008) were purchased from Thermo Fisher Scientific, Waltham, MA, USA. 10X Tris/Glycine buffer (161–0771), 10X Tris/Glycine/SDS buffer (161–0772), 2X Laemmli sample buffer (161–0737), and Protein Ladder (26616) were purchased from BioRad, CA, USA. Anti- FOXO3a Polyclonal Antibody (PA5-27145), anti-SIRT1 Antibody (MA5-15677), and Anti-Actin Antibody (MA1-744) were purchased from Invitrogen and Thermo Fisher Scientific Waltham, MA, USA. Protease inhibitor (04,693,124 001), Fluorescence mounting media (S3023), 2-Mercaptoethanol (M6250), Phosphate buffered saline (P5368), and Paraformaldehyde (P6148) were purchased from Sigma Aldrich St. Louis, MO, USA.