Confluent EC were treated with DMSO or human TGF-β1 (R&D, 10ng/ml) for 24 hours. Cells were washed twice with ice-cold PBS and lysed using RIPA Biffer (Thermo Scientific, 25mM Tris•HCl pH 7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS).
30 μg of protein was separated using 8% SDS-PAGE and transferred to PVDF membrane (Bio-Rad). Membranes were incubated with the anti-TSP-4 (R&D); anti-TSP-1 (Thermo Fisher); anti-phospho-SMAD3, anti-phospho-SMAD2, anti-phospho-SMAD1/5/9, and anti-SMAD1 (Cell Signaling); anti-β-actin (Sigma) primary antibodies followed by horseradish peroxidase–linked IgG; and visualized by chemiluminescence using Super Signal West Pico Chemiluminescence substrate (Thermo Fisher).
Cells were pre-treated with 2.5 μM SIS3 (Calbiochem), 10 μM SB431542 (Cayman Chemical) and cyclohexamide (Sigma-Aldrich, 10 μg/ml) for 30 min before TGF-β1 stimulation.
30 μg of protein was separated using 8% SDS-PAGE and transferred to PVDF membrane (Bio-Rad). Membranes were incubated with the anti-TSP-4 (R&D); anti-TSP-1 (Thermo Fisher); anti-phospho-SMAD3, anti-phospho-SMAD2, anti-phospho-SMAD1/5/9, and anti-SMAD1 (Cell Signaling); anti-β-actin (Sigma) primary antibodies followed by horseradish peroxidase–linked IgG; and visualized by chemiluminescence using Super Signal West Pico Chemiluminescence substrate (Thermo Fisher).
Cells were pre-treated with 2.5 μM SIS3 (Calbiochem), 10 μM SB431542 (Cayman Chemical) and cyclohexamide (Sigma-Aldrich, 10 μg/ml) for 30 min before TGF-β1 stimulation.