Pierce coomassie plus protein assay

Extracellular proteins (ECPs) were precipitated from cell-free supernatants by ammonium sulfate precipitation, as previously described [30 (link), 64 (link)]. Briefly, ammonium sulfate crystals (Fisher Scientific) were added to cell-free supernatants to achieve 65% saturation and incubated at 4 °C with gentle mixing for 24 h. Precipitated proteins were collected by centrifugation, resuspended in Tris buffer, and dialyzed twice against the same buffer in 10 Kda dialysis cassettes (Slide-A-Lyzer (Thermo Fisher)). Following dialysis, the volume of all protein samples were adjusted to 20 ml by the addition of cold Tris buffer. Protein concentration of each sample was determined by the Bradford assay (Pierce Coomassie Plus Protein Assay, Thermo Fisher). These concentrated proteins were used for all enzymatic assays.

Whole cell lysates were prepared as previously described [17 (link)]. Total protein was measured using the Pierce Coomassie Plus Protein Assay (Thermo Fisher Scientific, Waltham, MA). For human bronchial epithelial cells, the volumes of whole cell lysate loaded per lane were adjusted to have similar Cytokeratin expression after 5 μl lysates were pre-run to measure Cytokeratin, because Cytokeratin confirms epithelial cells obtained by airway brushing. Equal amounts of protein (80 μg/lane) were loaded per lane for samples from BET1A cells, while Enolase was used as a loading control. Proteins were separated by electrophoresis on a 4–15% Tris-HCl precast gel (Bio-Rad Lab, Hercules, CA) and transferred onto polyvinylidene difluoride membranes (PVDF, Millipore Corporation, Bedford, MA). Rabbit anti-iNOS (sc-651, 1:400), ARG2 (sc-20151, 1:600) and Enolase (sc-15343, 1:1000) polyclonal Ab (Santa Cruz Biotechnology, Santa Cruz, CA), and mouse anti-Cytokeratin (M3515, 1:400) monoclonal Ab (DAKO North America, Carpinteria, California) were used in western analyses.

Supernatants of bursa-derived cells were tested for Col I secretion using the MicroVue CICP EIA Kit (Quidel, San Diego, CA, United States) for determining levels of the C-Terminal propeptide of Type I Collagen (CICP). CICP values were calculated from the standard curve after measuring the optical density at 405 nm with the Tecan spectrophotometer. For quantification of the MMP and TIMP secretion under mechanical stimulation, the concentrations of total MMP1, MMP2, MMP3, and TIMP1 were determined using commercially available sandwich ELISAs (DuoSet ELISA, R&D Systems, Wiesbaden, Germany) according to the manufacturer’s manual. Col I, MMP, and TIMP concentrations were normalized to the total protein concentration measured using Pierce Coomassie Plus protein assay (Thermo Fisher Scientific, Waltham, MA, United States).