Intercept blocking buffer

Nitrocellulose membranes containing the transferred proteins were blocked in Intercept Blocking Buffer (LI-COR Bioscience) for 1-hour at room temperature. Membranes were probed with primary antibodies, including mouse anti-mTOR (1:1000; Cat# 4517), mouse anti-Akt (1:1000; Cat# 2920), mouse anti-GSK3β (1:1000; Cat# 9832), mouse anti-ERK (1:1000; Cat# 9107), rabbit anti-phospho mTOR (1:1000; Cat# 5536 S), rabbit anti-phospho Akt (1:1000; Cat# 4060 S), rabbit anti-phospho GSK3β (1:1000; Cat# 5558), rabbit anti-phospho ERK (1:1000; Cat# 4376) (all antibodies were purchased from Cell Signaling Technologies) diluted in Intercept Blocking Buffer + 0.2% Tween-20 and incubated overnight at 4 °C, before being exposed to secondary antibody (IRDye^® 680RD Donkey anti-Mouse IgG and IRDye^® 800CW Donkey anti-Rabbit IgG) (LI-COR Biosciences) for 1-hour at room temperature in the dark.
Membranes were scanned on the LI-COR Bioscience Odyssey CLx imaging system and imaged using LI-COR Image Studio software version 2.1.10. All densitometry analyses were performed using Image Studio Light version 5. The region of interest encircling each band was defined automatically. All bands at the correct molecular weight were analyzed as the signal for that target protein. Values for each protein were normalized to loading control β-tubulin (Abcam).

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.

Western blotting was used to assess the presence of EV and non-EV protein markers. Equal protein amounts of EV samples were mixed with 4X Laemmli buffer (Bio-Rad), denatured for 5 min at 95 °C, and loaded onto 4–20% Mini-PROTEAN TGX Stain-Free Protein Gels (Bio-Rad). SDS-PAGE was run for 1.5 h at 90 V; then, proteins were transferred to nitrocellulose membranes (Cytiva) at 100 V for 1 h. Membranes were blocked with LI-COR Intercept Blocking Buffer (LI-COR Biosciences, Bad Homburg, Germany) for 1 h at RT. Blocked membranes were incubated overnight at 4 °C with primary antibodies diluted in LI-COR blocking buffer with 0.1% Tween-20. Membranes were washed with TBS-T (TBS with 0.1% Tween-20) three times for 5 min and then incubated with secondary antibodies for 1 h at RT. Incubation was followed by three additional washes with TBS-T of 5 min each. Blots were imaged using the Odyssey Infrared Imaging System (LI-COR Biosciences). The detailed list of primary and secondary antibodies used is provided in Supplementary Table S5. Original images shown in File S1.

Western blotting was used to assess the presence of EV and non-EV protein markers. Equal protein amounts of EV samples were mixed with 4X Laemmli buffer (Bio-Rad, Herculaes, CA, USA), denatured for 5 min at 95 °C, and loaded onto 4–20% Mini-PROTEAN TGX Stain-Free Protein Gels (Bio-Rad). SDS-PAGE was run for 1.5 h at 90 V, and then proteins were transferred to nitrocellulose membranes (Cytiva) at 100 V for 1 h. Membranes were blocked with LI-COR Intercept Blocking Buffer (LI-COR Biosciences) for 1 h at RT. Blocked membranes were incubated overnight at 4°C with primary antibodies diluted in LI-COR blocking buffer with 0.1% Tween-20. Membranes were washed with TBS-T (TBS with 0.1% Tween-20) 3 times for 5 min, and then incubated with secondary antibodies for 1 h at RT. Incubation was followed by 3 additional washes with TBS-T, 5 min each. Blots were imaged using the Odyssey Infrared Imaging System (LI-COR Biosciences). The detailed list of primary and secondary antibodies used is provided in Supplementary Table S1.