Anti rabbit horseradish peroxidase hrp conjugated secondary antibody

Equal amounts of protein (30 µg) were mixed with NuPAGE LDS Sample Buffer (Thermo Scientific) and denatured at 70 °C for 10 min. After denaturation, NuPAGE Reducing Agent (Thermo Scientific) was added. Next, the samples were separated by SDS-PAGE, followed by wet protein transfer to methanol activated Novex® pre-cut polyvinylidene difluoride membranes (Thermo Scientific). The membrane was blocked for 1 h at room temperature with 5% nonfat dry milk in TBS/0.2% Tween-20 (TBST) to prevent nonspecific binding. Then, the membrane was washed (three times for 5 min) in TBST followed by incubation with rabbit monoclonal anti-BMP-6 (Abcam) in 5% BSA in TBST overnight at 4 °C while shaking. After washing (three times for 5 min), the membrane was incubated with an anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (GE Healthcare) in 5% nonfat dry milk in TBST for 1 h at room temperature. The proteins were visualized with SuperSignal™ West Dura Extended Duration Substrate (Thermo Scientific) and documented by the ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA). Band quantification was performed in Image Lab (version 3.0, Bio-Rad, Hercules, CA). ELISA CCL2 and TGF-β1 protein levels in the BAL fluid were determined by an ELISA kit purchased from R&D Systems (Abingdon, UK). All ELISAs were performed following the manufacturer's instructions.