Rabbit anti phospho smad3

Lung tissues or cells were lysed in RIPA buffer containing complete protease inhibitor and PhosSTOP phosphatase inhibitor cocktails (Roche Applied Science). Lysate was centrifuged at 13,500 × g for 10 min at 4 °C, and the supernatant was collected. Protein concentrations were determined by the BCA assay (Pierce). Laemmli buffer (Bio-Rad) containing β-mercaptoethanol was added to 40 μg protein, and boiled at 95 °C for 5 min. Protein were separated by SDS–PAGE, transferred to nitrocellulose membranes (Millipore) overnight, blocked with 5% bovine serum albumin in PBS containing 0.05% Tween-20 for 1 h and incubated with primary antibody overnight at 4 °C. Primary antibodies used were: rabbit anti-KLF4, rabbit anti-SMAD2/3, rabbit anti-GAPDH (1:500, 1:500, 1:5000; Cell Signaling Technology), rabbit anti-phospho-SMAD3, rabbit anti-collagen 1, rabbit anti-CCL2, rabbit anti-FOXM1 (1:500, 1:1000, 1:1000, 1:1000; Abcam), mouse anti-SMA (1:2000, Sigma) and mouse anti-fibronectin (1:5000, BD Biosciences). After washing, membranes were incubated with horseradish peroxidase–conjugated secondary antibodies (1:2000; DAKO) for 1 h. Detection was performed with the Western Blotting Substrate Plus (Pierce) and GBOX imaging system (Syngene).